ANGIOTENSINOGEN (AGT) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the angiotensinogen (AGT) gene, and methods of using such RNAi agents to inhibit expression of AGT and methods of treating subjects having an AGT-associated disorder, e.g., hypertension.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/274,393, filed on Feb. 13, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/313,145, filed on Nov. 22, 2016, now U.S. Pat.No. 10,238,749, issued on Mar. 26, 2019, which is a 35 U.S. C. § 371National Stage Filing of International Application No.PCT/US2015/032099, filed on May 22, 2015, which claims the benefit ofand priority to U.S. Provisional Application No. 62/001,731, filed onMay 22, 2014, and U.S. Provisional Application No. 62/047,978, filed onSep. 9, 2014. The entire contents of each of the foregoing applicationsare hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incoroporated byreference in its entirety. The ASCII copy, created on Aug. 17, 2020 isnamed 121301 01305 SL.txt and is 318,809 bytes in size.

BACKGROUND OF THE INVENTION

The renin-angiotensin-aldosterone system (RAAS) plays a crucial role inthe regulation of blood pressure. The RAAS cascade begins with therelease of renin by the juxtaglomerular cells of the kidney into thecirculation. Renin secretion is stimulated by several factors, includingNa+ load in the distal tubule, β-sympathetic stimulation, and/or reducedrenal perfusion. Active renin in the plasma cleaves angiotensinogen(produced by the liver) to angiotensin I, which is then converted bycirculating and locally expressed angiotensin-converting enzyme (ACE) toangiotensin II. Most of the effects of angiotensin II on the RAAS areexerted by its binding to angiotensin II type 1 receptors (AT₁R),leading to arterial vasoconstriction, tubular and glomerular effects,such as enhanced Na+ reabsorption or modulation of glomerular filtrationrate. In addition, together with other stimuli such asadrenocorticotropin, anti-diuretic hormone, catecholamines, endothelin,serotonin, and levels of Mg2+ and K+, AT₁R stimulation leads toaldosterone release which, in turn, promotes Na+ and K+ excretion in therenal distal convoluted tubule.

Dysregulation of the RAAS leading to, for example, excessive angiotensinII production and/or AT₁R stimulation results in hypertension which canlead to, e.g., increased oxidative stress, promotion of inflammation,hypertrophy, and fibrosis in the heart, kidneys, and arteries, andresult in, e.g., left ventricular fibrosis, arterial remodeling, andglomerulosclerosis.

Hypertension is the most prevalent, controllable disease in developedcountries, affecting 20-50% of adult populations. It is a major riskfactor for various diseases, disorders and conditions such as, shortenedlife expectancy, chronic kidney disease, stroke, myocardial infarction,heart failure, aneurysms (e.g. aortic aneurysm), peripheral arterydisease, heart damage (e.g., heart enlargement or hypertrophy) and othercardiovascular related diseases, disorders and/or conditions. Inaddition, hypertension has been shown to be an important risk factor forcardiovascular morbidity and mortality accounting for, or contributingto, 62% of all strokes and 49% of all cases of heart disease.

Despite the number of anti-hypertensive drugs available for treatinghypertension, more than two-thirds of subjects are not controlled withone anti-hypertensive agent and require two or more anti-hypertensiveagents selected from different drug classes. This further reduces thenumber of subjects with controlled blood pressure as compliance andside-effects increase with increasing medication.

Accordingly, there is a need in the art for alternative therapies andcombination therapies for subjects having an angiotensinogen-associateddisease.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an angiotensinogen (AGT) gene. The AGT gene may be withina cell, e.g., a cell within a subject, such as a human.

The present invention also provides methods and therapies for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an AGT gene, e.g., an angiotensinogen-associateddisease, such as hypertension, using iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an AGT gene for inhibiting the expression of an AGT gene.

Accordingly, in one aspect, the present invention providesdouble-stranded ribonucleic acid (RNAi) agents for inhibiting expressionof angiotensinogen (AGT), which comprise a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:1 and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:2, wherein substantially all of the nucleotides of the sense strandand substantially all of the nucleotides of the antisense strand aremodified nucleotides, and wherein the sense strand is conjugated to aligand attached at the 3′-terminus. In one embodiment, all of thenucleotides of the sense strand and all of the nucleotides of theantisense strand are modified nucleotides.

In another aspect, the present invention provides double-strandedribonucleic acid (RNAi) agents for inhibiting expression ofangiotensinogen (AGT), which comprise a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from nucleotides 2801-2101; 803-843; 834-859; 803-859;803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624;1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223;2227-2360; 2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342;2333-2360; 2333-2358; 485-503; 517-535; 560-578; 635-653; 803-821;814-832; 822-840; 825-843; 834-852; 837-855; 841-859; 855-873; 967-985;1247-1265; 1248-1266; 1249-1267; 1251-1269; 1253-1271; 1566-1584;1570-1588; 1572-1590; 1574-1592; 1584-1602; 1587-1605; 1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747;1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845;1880-1989; 1892-1914; 1894-1914; 1894-2012; 2035-2053; 2046-2064;2057-2075; 2070-2088; 2072-2090; 2078-2096; 2078-2107; 2078-2011;2080-2098; 2081-2099; 2081-2104; 2081-2011; 2082-2100; 2084-2102;2084-2011; 2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143;2167-2185; 2179-2197; 2201-2219; 2202-2220; 2203-2221; 2204-2222;2227-2245; 2230-2248; 2234-2252; 2244-2264; 2255-2273; 2266-2284;2268-2286; 2270-2288; 2279-2297; 2281-2299; 2283-2301; 2284-2302;2285-2303; 2286-2304; 2288-2306; 2290-2308; 2291-2309; 2291-2311;2291-2318; 2291-2315; 2292-2310; 2294-2312; 2296-2314; 2299-2317;2304-2322; 2304-2329; 2306-2324; 2307-2325; 2309-2327; 2309-2329;2309-2342; 2309-2350; 2309-2358; 2314-2332; 2316-2334; 2317-2335;2320-2338; 2321-2339; 2323-2341; 2325-2343; 2326-2344; 2328-2346;2329-2347; 2331-2349; 2333-2351; 2334-2352; 2335-2353; 2339-2357;2340-2358; or 2341-2359 of the nucleotide sequence of SEQ ID NO:1 andthe antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotides at thecorresponding position of the nucleotide sequence of SEQ ID NO:2 suchthat the antisense strand is substantially complementary to the at least15 contiguous nucleotides in the sense strand. In certain embodiments,substantially all of the nucleotides of the sense strand are modifiednucleotides. In other embodiments, substantially all of the nucleotidesof the antisense strand are modified nucleotides. In yet otherembodiments, substantially all of the nucleotides of both strands aremodified nucleotides. In one embodiment, all of the nucleotides of thesense strand and all of the nucleotides of the antisense strand aremodified nucleotides. In one embodiment, the sense strand is conjugatedto a ligand attached at the 3′-terminus. In one embodiment, the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from nucleotides 2801-2101 of the nucleotide sequenceof SEQ ID NO:1 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesat the corresponding position of the nucleotide sequence of SEQ ID NO:2such that the antisense strand is substantially complementary to the atleast 15 contiguous nucleotides in the sense strand.

In one aspect, the present invention provides double-strandedribonucleic acid (RNAi) agents for inhibiting expression ofangiotensinogen (AGT), which comprise a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides from nucleotides 803-843;834-859; 803-859; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624;1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360;2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333-2360;2333-2358; 485-503; 517-535; 560-578; 635-653; 803-821; 814-832;822-840; 825-843; 834-852; 837-855; 841-859; 855-873; 967-985;1247-1265; 1248-1266; 1249-1267; 1251-1269; 1253-1271; 1566-1584;1570-1588; 1572-1590; 1574-1592; 1584-1602; 1587-1605; 1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747;1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845;1880-1989; 1894-2012; 2035-2053; 2046-2064; 2057-2075; 2070-2088;2072-2090; 2078-2096; 2080-2098; 2081-2099; 2082-2100; 2084-2102;2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143; 2167-2185;2179-2197; 2201-2219; 2202-2220; 2203-2221; 2204-2222; 2227-2245;2230-2248; 2234-2252; 2244-2264; 2255-2273; 2266-2284; 2268-2286;2270-2288; 2279-2297; 2281-2299; 2283-2301; 2284-2302; 2285-2303;2286-2304; 2288-2306; 2290-2308; 2291-2309; 2292-2310; 2294-2312;2296-2314; 2299-2317; 2304-2322; 2306-2324; 2307-2325; 2309-2327;2314-2332; 2316-2334; 2317-2335; 2320-2338; 2321-2339; 2323-2341;2325-2343; 2326-2344; 2328-2346; 2329-2347; 2331-2349; 2333-2351;2334-2352; 2335-2353; 2339-2357; 2340-2358; or 2341-2359 of thenucleotide sequence of SEQ ID NO:1 and the antisense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleoidesfrom the nucleotides at the corresponding position of the nucleotidesequence of SEQ ID NO:2 such that the antisense strand is substantiallycomplementary to the at least 15 contiguous nucleotides in the sensestrand. In certain embodiments, substantially all of the nucleotides ofthe sense strand are modified nucleotides. In other embodiments,substantially all of the nucleotides of the antisense strand aremodified nucleotides. In yet other embodiments, substantially all of thenucleotides of both strands are modified nucleotides. In one embodiment,the sense strand is conjugated to a ligand attached at the 3′-terminus.

In one embodiment, the sense strand and the antisense strand comprise aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of theantisense sequences listed in any one of Tables 3, 4, 7, 8, 11, 13, and15. For example, in certain embodiments, the sense strand and theantisense strand comprise a region of complementarity which comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the antisense sequences of the duplexes AD-52433.1,AD-52438.1, AD-52439.1, AD-52445.1, AD-52449.1, AD-52451.1, AD-52456.1,AD-52457.1, AD-52462.1, AD-52463.1, AD-52469.1, AD-52474.1, AD-55976.1,AD-55978.1, AD-55979.1, AD-55980.1, AD-55981.1, AD-55982.1, AD-55983.1,AD-55984.1, AD-55987.1, AD-55988.1, AD-55989.1, AD-55990.1, AD-55991.1,AD-55994.1, AD-55995.1, AD-55996.1, AD-55999.1, AD-56000.1, AD-56001.1,AD-56002.1, AD-56003.1, AD-56006.1, AD-56007.1, AD-56008.1, AD-56009.1,AD-56011.1, AD-56012.1, AD-56013.1, AD-56016.1, AD-56017.1, AD-56019.1,AD-56020.1, AD-56021.1, AD-56022.1, AD-56024.1, AD-56026.1, AD-56027.1,AD-56029.1, AD-56030.1, AD-56031.1, AD-56032.1, AD-56035.1, AD-56039.1,AD-56041.1, AD-56043.1, AD-56044.1, AD-56047.1, AD-56048.1, AD-56051.1,AD-56053.1, AD-56054.1, AD-56059.1, AD-56062.1, AD-56065.1, AD-56066.1,AD-60770.1, AD-60771.1, AD-60776.1, AD-60777.1, AD-60778.1, AD-60779.1,AD-60780.1, AD-60781.1, AD-60783.1, AD-60784.1, AD-60785.1, AD-60788.1,AD-60789.1, AD-60791.1, AD-60793.1, AD-60795.1, AD-60798.1, AD-60801.1,AD-67903.1, AD-67906.1, AD-67923.1, AD-67924.1, AD-67925.1, AD-67926.1,AD-67935.1, AD-67965.1, AD-67994.1, AD-67995.1, AD-67996.1, AD-68017.1,AD-68022.1, AD-68035.1, AD-68036.1, AD-68037.1, AD-68084.2, AD-68085.2,AD-68086.2, AD-68087.2, AD-68090.2, AD-68091.2, AD-68092.2, AD-68093.2,AD-68116.1, AD-68117.1, AD-68118.1, AD-68124.1, AD-68125.1, orAD-68126.1. In certain embodiments, the sense strand and the antisensestrand comprise a region of complementarity which comprises at least 15contiguous nucleotides of the region of complementarity of any one ofduplexes AD-52433.1, AD-52438.1, AD-52439.1, AD-52445.1, AD-52449.1,AD-52451.1, AD-52456.1, AD-52457.1, AD-52462.1, AD-52463.1, AD-52469.1,AD-52474.1, AD-55976.1, AD-55978.1, AD-55979.1, AD-55980.1, AD-55981.1,AD-55982.1, AD-55983.1, AD-55984.1, AD-55987.1, AD-55988.1, AD-55989.1,AD-55990.1, AD-55991.1, AD-55994.1, AD-55995.1, AD-55996.1, AD-55999.1,AD-56000.1, AD-56001.1, AD-56002.1, AD-56003.1, AD-56006.1, AD-56007.1,AD-56008.1, AD-56009.1, AD-56011.1, AD-56012.1, AD-56013.1, AD-56016.1,AD-56017.1, AD-56019.1, AD-56020.1, AD-56021.1, AD-56022.1, AD-56024.1,AD-56026.1, AD-56027.1, AD-56029.1, AD-56030.1, AD-56031.1, AD-56032.1,AD-56035.1, AD-56039.1, AD-56041.1, AD-56043.1, AD-56044.1, AD-56047.1,AD-56048.1, AD-56051.1, AD-56053.1, AD-56054.1, AD-56059.1, AD-56062.1,AD-56065.1, AD-56066.1, AD-60770.1, AD-60771.1, AD-60776.1, AD-60777.1,AD-60778.1, AD-60779.1, AD-60780.1, AD-60781.1, AD-60783.1, AD-60784.1,AD-60785.1, AD-60788.1, AD-60789.1, AD-60791.1, AD-60793.1, AD-60795.1,AD-60798.1, AD-60801.1, AD-67903.1, AD-67906.1, AD-67923.1, AD-67924.1,AD-67925.1, AD-67926.1, AD-67935.1, AD-67965.1, AD-67994.1, AD-67995.1,AD-67996.1, AD-68017.1, AD-68022.1, AD-68035.1, AD-68036.1, AD-68037.1,AD-68084.2, AD-68085.2, AD-68086.2, AD-68087.2, AD-68090.2, AD-68091.2,AD-68092.2, AD-68093.2, AD-68116.1, AD-68117.1, AD-68118.1, AD-68124.1,AD-68125.1, or AD-68126.1.

In one embodiment, the antisense strand comprises a region ofcomplementarity which comprises at least 15 contiguous unmodifiednucleotides differing by no more than 3 nucleotides from the antisensenucleotide sequence of AD-67327 (5′-AUUAGAAGAAAAGGUGGGAGACU-3′; SEQ IDNO:537). In another embodiment, the region of complementarity consistsof the antisense unmodified nucleotide sequence of AD-67327(5′-AUUAGAAGAAAAGGUGGGAGACU-3′; SEQ ID NO:537). In one embodiment, thedsRNA comprises a sense strand consisting of the nucleotide sequence of5′-UCUCCCACCUUUUCUUCUAAU-3′ (SEQ ID NO:499), and an antisense strandconsisting of the nucleotide sequence of 5′-UUAGAAGAAAAGGUGGGAGACU-3′(SEQ ID NO:537). In one embodiment, the double-stranded RNAi agentcomprises the modified nucleotide sequence of AD-67327(5′-uscsucccAfcCfUfUfuucuucuaau-3′; SEQ ID NO:1037 and5′-asUfsuagAfagaaaagGfuGfggagascsu-3′; SEQ ID NO:1038.

In some embodiments, the modified nucleotide(s) is independentlyselected from the group consisting of a 2′-O-methyl modified nucleotide,a 2′-fluoro modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or a dodecanoic acid bisdecylamide group. Infurther embodiments, the modified nucleotide is selected from the groupconsisting of a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an unlockednucleotide a conformationally restricted nucleotide, a contrained ethylnucleotide, an a basic nucleotide, a 2′-amino-modified nucleotide, a2′-alkyl-modified nucleotide, a 2′-O-allyl modified nucleotide, a2′-C-allyl modified nucleotide, a 2′-hydroxyl modified nucleotide, amorpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide.

In another embodiment of the double-stranded RNAi agent, at least onestrand comprises a 3′ overhang of at least 1 nucleotide. In anotherembodiment, at least one strand comprises a 3′ overhang of at least 2nucleotides.

In another aspect, the present invention provides RNAi agents, e.g.,double-stranded ribonucleic acid (RNAi) agents capable of inhibiting theexpression of angiotensinogen (AGT) in a cell, wherein thedouble-stranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding AGT, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double-strandedRNAi agent is represented by formula (III):

sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′r-N_(a)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and 1 are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof;

each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0;or both i and j are 1.

In another embodiment, k is 0; 1 is 0; k is 1; 1 is 1; both k and 1 are0; or both k and l are 1.

In one embodiment, XXX is complementary to X′X′X′, YYY is complementaryto Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand.

In another embodiment, the Y′Y′Y′ motif occurs at the 11, 12 and 13positions of the antisense strand from the 5′-end.

In one embodiment, Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (Ma):

sense: 5′n _(p)—N_(a)—YYY—N_(a)-n _(q)3′

antisense: 3′n _(p)′—N_(a)′—Y′Y′Y′—N_(a)′-n _(q)′5′  (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):

sense: 5′n _(p)—N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′

antisense: 3′n _(p)′—N_(a)′—N_(a)′-n _(q)′5′  (IIIb)

-   -   wherein each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 1-5 modified nucleotides.

In yet another embodiment, formula (III) is represented by formula(IIIc):

sense: 5′n _(p)—N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′

antisense: 3′n _(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n_(q)′5′  (IIIc)

-   -   wherein each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 1-5 modified nucleotides.

In a further embodiment, formula (III) is represented by formula (IIId):

sense: 5′n _(p)—N_(a)—X X X—N_(b)—Y Y Y—N_(b)—Z Z Z—N_(a)-n _(q)3′

antisense: 3′n _(p)′—N_(a)′—X′X′X′—N_(a)′-n _(q′)5′   (IIId)

-   -   wherein each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 1-5 modified nucleotides and        each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 2-10 modified nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairsin length. In another embodiment, the double-stranded region is 17-23nucleotide pairs in length. In yet another embodiment, thedouble-stranded region is 17-25 nucleotide pairs in length. In a furtherembodiment, the double-stranded region is 23-27 nucleotide pairs inlength. In another embodiment, the double-stranded region is 19-21nucleotide pairs in length. In another embodiment, the double-strandedregion is 19-23 nucleotide pairs in length. In another embodiment, thedouble-stranded region is 21-23 nucleotide pairs in length. In yetanother embodiment, each strand has 15-30 nucleotides. In yet anotherembodiment, each strand has 19-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of LNA, UNA, CRN, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro,2′-deoxy, 2′-hydroxyl, and combinations thereof. In another embodiment,the modifications on the nucleotides are 2′-O-methyl or 2′-fluoromodifications.

In one embodiment, the ligand the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker. Inanother embodiment, the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sensestrand.

In another embodiment, the RNAi agent is conjugated to the ligand asshown in the following schematic

wherein X is O or S. In a specific embodiment, X is O.

In one embodiment, the agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.

In a further embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In anotherembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In anotherembodiment, the strand is the antisense strand. In a further embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In another embodiment, the strand is the antisense strand.

In another embodiment, the double-stranded RNAi agent comprises 6-8phosphorothioate internucleotide linkages. In a further embodiment, theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus of the sense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of theantisense strand of the duplex is an AU base pair. In anotherembodiment, the Y nucleotides contain a 2′-fluoro modification. In afurther embodiment, the Y′ nucleotides contain a 2′-O-methylmodification.

In one embodiment, p′>0. In another embodiment, p′=2. In a furtherembodiment, q′=0, p=0, q=0, and p′ overhang nucleotides arecomplementary to the target mRNA. In yet a further embodiment, q′=0,p=0, q=0, and p′ overhang nucleotides are non-complementary to thetarget mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In another embodiment, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage. In a further embodiment, alln_(p)′ are linked to neighboring nucleotides via phosphorothioatelinkages.

In another embodiment, the RNAi agent is selected from the group of RNAiagents listed in any one of Tables 3, 4, 7, 8, 11, 13, and 15.

In one aspect, the present invention provides double-strandedribonucleic acid (RNAi) agents for inhibiting expression ofangiotensinogen (AGT). The double-stranded RNAi agents include a sensestrand and an antisense strand forming a double-stranded region, whereinthe sense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:land the antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:2, wherein substantially all of the nucleotides of the sensestrand comprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification wherein the sensestrand comprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and wherein the sensestrand is conjugated to one or more GalNAc derivatives attached througha branched bivalent or trivalent linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In another aspect, the present invention provides RNAi agents, e.g.,double-stranded ribonucleic acid (RNAi) agents capable of inhibiting theexpression of AGT (angiotensinogen) in a cell, wherein thedouble-stranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding AGT, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double-strandedRNAi agent is represented by formula (III):

sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

-   -   wherein:    -   i, j, k, and 1 are each independently 0 or 1;    -   p, p′, q, and q′ are each independently 0-6;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may        not be present independently represents an overhang nucleotide;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand.

In yet another aspect, the present invention provides RNAi agents, e.g.,double-stranded ribonucleic acid (RNAi) agents capable of inhibiting theexpression of angiotensinogen (AGT) in a cell, wherein thedouble-stranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding AGT, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double-strandedRNAi agent is represented by formula (III):

sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

-   -   wherein:    -   i, j, k, and l are each independently 0 or 1;    -   each n_(p), n_(q), and n_(q)′, each of which may or may not be        present, independently represents an overhang nucleotide;    -   p, q, and q′ are each independently 0-6;    -   n_(p)′>0 and at least one n_(p)′ is linked to a neighboring        nucleotide via a phosphorothioate linkage;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand.

In a further aspect, the present invention provides RNAi agents, e.g.,double-stranded ribonucleic acid (RNAi agent) capable of inhibiting theexpression of angiotensinogen (AGT) in a cell, wherein thedouble-stranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding AGT, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double-strandedRNAi agent is represented by formula (III):

sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

-   -   wherein:    -   i, j, k, and 1 are each independently 0 or 1;    -   each n_(p), n_(q), and n_(q)′, each of which may or may not be        present, independently represents an overhang nucleotide;    -   p, q, and q′ are each independently 0-6;    -   n_(p)′>0 and at least one n_(p)′ is linked to a neighboring        nucleotide via a phosphorothioate linkage;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand,        wherein the ligand is one or more GalNAc derivatives attached        through a bivalent or trivalent branched linker.

In another aspect, the present invention provides RNAi agents, e.g.,double-stranded ribonucleic acid (RNAi) agents capable of inhibiting theexpression of angiotensinogen (AGT) in a cell, wherein thedouble-stranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding AGT, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double-strandedRNAi agent is represented by formula (III):

sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

-   -   wherein:    -   i, j, k, and l are each independently 0 or 1;    -   each n_(p), n_(q), and n_(q)′, each of which may or may not be        present, independently represents an overhang nucleotide;    -   p, q, and q′ are each independently 0-6;    -   n_(p)′>0 and at least one n_(p)′ is linked to a neighboring        nucleotide via a phosphorothioate linkage;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′;    -   wherein the sense strand comprises at least one phosphorothioate        linkage; and    -   wherein the sense strand is conjugated to at least one ligand,        wherein the ligand is one or more GalNAc derivatives attached        through a bivalent or trivalent branched linker.

In yet another aspect, the present invention provides RNAi agents, e.g.,double-stranded ribonucleic acid (RNAi) agent capable of inhibiting theexpression of angiotensinogen (AGT) in a cell, wherein thedouble-stranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding AGT, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double-strandedRNAi agent is represented by formula (III):

sense: 5′n _(p)—N_(a)—YYY—N_(a)-n _(q)3′

antisense: 3′n _(p)′—N_(a)′— Y′Y′Y′—N_(a)′-n _(q)′5′  (IIIa)

-   -   wherein:    -   each n_(p), n_(q), and n_(q)′, each of which may or may not be        present, independently represents an overhang nucleotide;    -   p, q, and q′ are each independently 0-6;    -   n_(p)′>0 and at least one n_(p)′ is linked to a neighboring        nucleotide via a phosphorothioate linkage;    -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   YYY and Y′Y′Y′ each independently represent one motif of three        identical modifications on three consecutive nucleotides, and        wherein the modifications are 2′-O-methyl or 2′-fluoro        modifications;    -   wherein the sense strand comprises at least one phosphorothioate        linkage; and    -   wherein the sense strand is conjugated to at least one ligand,        wherein the ligand is one or more GalNAc derivatives attached        through a bivalent or trivalent branched linker.

In another aspect, the present invention provides a double-stranded RNAiagent comprising the RNAi agents listed in any one of Tables 3, 4, 7, 8,11, 13, and 15.

In another aspect, the present invention provides a compositioncomprising a modified antisense polynucleotide agent, wherein the agentis capable of inhibiting the expression of angiotensinogen (AGT) in acell, and comprises a sequence complementary to a sense sequenceselected from the group of the sequences listed in Tables 3, 4, 7, 8,11, 13, and 15, wherein the polynucleotide is about 14 to about 30nucleotides in length.

The present invention also provides cells, vectors, host cells andpharmaceutical compositions comprising the double-stranded RNAi agentsof the invention.

In one embodiment, a cell contains the double-stranded RNAi agent.

In some embodiments, the double-stranded RNAi agent or the compositioncomprising a modified antisense polynucleotide agent is administeredusing a pharmaceutical composition.

In one embodiment, the pharmaceutical compositions of the inventioncomprise a lipid formulation, such as XTC or MC3.

In preferred embodiments, the double-stranded RNAi agent is administeredin a solution. In some embodiments, the double-stranded RNAi agent isadministered in an unbuffered solution. In another embodiment, theunbuffered solution is saline or water. In another embodiment, thedouble-stranded RNAi agent is administered with a buffer solution. Inyet another embodiment, the buffer solution comprises acetate, citrate,prolamine, carbonate, or phosphate or any combination thereof. In someembodiment, the buffer solution is phosphate buffered saline (PBS).

In another aspect, the present invention provides methods of inhibitingangiotensinogen (AGT) expression in a cell. The methods includecontacting the cell with the double-stranded RNAi agent, apharmaceutical composition, a composition comprising a modifiedantisense polynucleotide agent, or a vector comprising the RNAi agentand maintaining the cell produced for a time sufficient to obtaindegradation of the mRNA transcript of a AGT gene, thereby inhibitingexpression of the AGT gene in the cell.

In one embodiment, the cell is within a subject. In a furtherembodiment, the subject is a human. In a further embodiment, the subjectsuffers from an angiotensinogen-associated disease.

In one embodiment, the AGT expression is inhibited by at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 98% or about 100%.

In one embodiment, the sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from nucleotides2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875;1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624; 1584-1621;2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360; 2227-2304;2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333-2360; 2333-2358;485-503; 517-535; 560-578; 635-653; 803-821; 814-832; 822-840; 825-843;834-852; 837-855; 841-859; 855-873; 967-985; 1247-1265; 1248-1266;1249-1267; 1251-1269; 1253-1271; 1566-1584; 1570-1588; 1572-1590;1574-1592; 1584-1602; 1587-1605; 1591-1609; 1592-1610; 1595-1613;1601-1619; 1602-1620; 1605-1623; 1729-1747; 1738-1756; 1739-1757;1741-1769; 1767-1785; 1810-1828; 1827-1845; 1880-1989; 1892-1914;1894-1914; 1894-2012; 2035-2053; 2046-2064; 2057-2075; 2070-2088;2072-2090; 2078-2096; 2078-2107; 2078-2011; 2080-2098; 2081-2099;2081-2104; 2081-2011; 2082-2100; 2084-2102; 2084-2011; 2090-2108;2100-2118; 2111-2129; 2124-2142; 2125-2143; 2167-2185; 2179-2197;2201-2219; 2202-2220; 2203-2221; 2204-2222; 2227-2245; 2230-2248;2234-2252; 2244-2264; 2255-2273; 2266-2284; 2268-2286; 2270-2288;2279-2297; 2281-2299; 2283-2301; 2284-2302; 2285-2303; 2286-2304;2288-2306; 2290-2308; 2291-2309; 2291-2311; 2291-2318; 2291-2315;2292-2310; 2294-2312; 2296-2314; 2299-2317; 2304-2322; 2304-2329;2306-2324; 2307-2325; 2309-2327; 2309-2329; 2309-2342; 2309-2350;2309-2358; 2314-2332; 2316-2334; 2317-2335; 2320-2338; 2321-2339;2323-2341; 2325-2343; 2326-2344; 2328-2346; 2329-2347; 2331-2349;2333-2351; 2334-2352; 2335-2353; 2339-2357; 2340-2358; or 2341-2359 ofthe nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention provides methods of treating asubject having a angiotensinogen (AGT)-associated disorder, comprisingadministering to the subject a therapeutically effective amount of thedouble-stranded RNAi agent, a composition comprising a modifiedantisense polynucleotide agent, or a pharmaceutical compositioncomprising the double-stranded RNAi agent, thereby treating the subject.

In another aspect, the present invention provides methods of treating asubject having a angiotensinogen (AGT)-associated disorder which includesubcutaneously administering to the subject a therapeutically effectiveamount of a double-stranded ribonucleic acid (RNAi agent), wherein thedouble-stranded RNAi agent comprises a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:1 and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:2, wherein substantially all of the nucleotides of the antisensestrand comprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoromodification, wherein theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, wherein substantially all of the nucleotides of thesense strand comprise a modification selected from the group consistingof a 2′-O-methyl modification and a 2′-fluoro modification, wherein thesense strand comprises two phosphorothioate internucleotide linkages atthe 5′-terminus and, wherein the sense strand is conjugated to one ormore GalNAc derivatives attached through a branched bivalent ortrivalent linker at the 3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In another aspect, the present invention provides methods of treating asubject having a angiotensinogen (AGT)-associated disorder which includeadministering to the subject a therapeutically effective amount of adouble-stranded ribonucleic acid (RNAi agent), wherein thedouble-stranded RNAi agent comprises a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from nucleotides 2801-2101; 803-843; 834-859; 803-859;803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624;1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223;2227-2360; 2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342;2333-2360; 2333-2358; 485-503; 517-535; 560-578; 635-653; 803-821;814-832; 822-840; 825-843; 834-852; 837-855; 841-859; 855-873; 967-985;1247-1265; 1248-1266; 1249-1267; 1251-1269; 1253-1271; 1566-1584;1570-1588; 1572-1590; 1574-1592; 1584-1602; 1587-1605; 1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747;1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845;1880-1989; 1892-1914; 1894-1914; 1894-2012; 2035-2053; 2046-2064;2057-2075; 2070-2088; 2072-2090; 2078-2096; 2078-2107; 2078-2011;2080-2098; 2081-2099; 2081-2104; 2081-2011; 2082-2100; 2084-2102;2084-2011; 2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143;2167-2185; 2179-2197; 2201-2219; 2202-2220; 2203-2221; 2204-2222;2227-2245; 2230-2248; 2234-2252; 2244-2264; 2255-2273; 2266-2284;2268-2286; 2270-2288; 2279-2297; 2281-2299; 2283-2301; 2284-2302;2285-2303; 2286-2304; 2288-2306; 2290-2308; 2291-2309; 2291-2311;2291-2318; 2291-2315; 2292-2310; 2294-2312; 2296-2314; 2299-2317;2304-2322; 2304-2329; 2306-2324; 2307-2325; 2309-2327; 2309-2329;2309-2342; 2309-2350; 2309-2358; 2314-2332; 2316-2334; 2317-2335;2320-2338; 2321-2339; 2323-2341; 2325-2343; 2326-2344; 2328-2346;2329-2347; 2331-2349; 2333-2351; 2334-2352; 2335-2353; 2339-2357;2340-2358; or 2341-2359 of the nucleotide sequence of SEQ ID NO:1 andthe antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotides at thecorresponding position of the nucleotide sequence of SEQ ID NO:2 suchthat the antisense strand is substantially complementary to the at least15 contiguous nucleotides in the sense strand. In certain embodiments,substantially all of the nucleotides of the sense strand are modifiednucleotides. In other embodiments, substantially all of the nucleotidesof the antisense strand are modified nucleotides. In yet otherembodiments, substantially all of the nucleotides of both strands aremodified nucleotides. In one embodiment, all of the nucleotides of thesense strand and all of the nucleotides of the antisense strand aremodified nucleotides. In one embodiment, the sense strand is conjugatedto a ligand attached at the 3′-terminus.

In another aspect, the present invention provides methods of treating asubject having a angiotensinogen (AGT)-associated disorder which includeadministering to the subject a therapeutically effective amount of adouble-stranded ribonucleic acid (RNAi agent), wherein thedouble-stranded RNAi agent comprises a sense strand and an antisensestrand forming a double-stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides from nucleotides 803-843;834-859; 803-859; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624;1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360;2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333-2360;2333-2358; 485-503; 517-535; 560-578; 635-653; 803-821; 814-832;822-840; 825-843; 834-852; 837-855; 841-859; 855-873; 967-985;1247-1265; 1248-1266; 1249-1267; 1251-1269; 1253-1271; 1566-1584;1570-1588; 1572-1590; 1574-1592; 1584-1602; 1587-1605; 1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747;1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845;1880-1989; 1894-2012; 2035-2053; 2046-2064; 2057-2075; 2070-2088;2072-2090; 2078-2096; 2080-2098; 2081-2099; 2082-2100; 2084-2102;2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143; 2167-2185;2179-2197; 2201-2219; 2202-2220; 2203-2221; 2204-2222; 2227-2245;2230-2248; 2234-2252; 2244-2264; 2255-2273; 2266-2284; 2268-2286;2270-2288; 2279-2297; 2281-2299; 2283-2301; 2284-2302; 2285-2303;2286-2304; 2288-2306; 2290-2308; 2291-2309; 2292-2310; 2294-2312;2296-2314; 2299-2317; 2304-2322; 2306-2324; 2307-2325; 2309-2327;2314-2332; 2316-2334; 2317-2335; 2320-2338; 2321-2339; 2323-2341;2325-2343; 2326-2344; 2328-2346; 2329-2347; 2331-2349; 2333-2351;2334-2352; 2335-2353; 2339-2357; 2340-2358; or 2341-2359 of thenucleotide sequence of SEQ ID NO:1 and the antisense strand comprises atleast 15 contiguous nucleotides from the nucleotides at thecorresponding position of the nucleotide sequence of SEQ ID NO:2 suchthat the antisense strand is substantially complementary to the at least15 contiguous nucleotides in the sense strand. In certain embodiments,substantially all of the nucleotides of the sense strand are modifiednucleotides. In other embodiments, substantially all of the nucleotidesof the antisense strand are modified nucleotides. In yet otherembodiments, substantially all of the nucleotides of both strands aremodified nucleotides. In one embodiment, the sense strand is conjugatedto a ligand attached at the 3′-terminus.

In one embodiment, the subject is a human.

In one embodiment, the angiotensinogen-associated disease is selectedfrom the group consisting of hypertension, borderline hypertension,primary hypertension, secondary hypertension, hypertensive emergency,hypertensive urgency, isolated systolic or diastolic hypertension,pregnancy-associated hypertension, diabetic hypertension, resistanthypertension, refractory hypertension, paroxysmal hypertension,renovascular hypertension, Goldblatt hypertension, ocular hypertension,glaucoma, pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy, diabetic nephropathy, diabeticretinopathy, chronic heart failure, cardiomyopathy, diabetic cardiacmyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurism,ventricular fibrosis, Cushing's syndrome, and other glucocorticoidexcess states including chronic steroid therapy, pheochromocytoma,reninoma, secondary aldosteronism and other mineralocorticoid excessstates, sleep apnea, thyroid/parathyroid disease, heart failure,myocardial infarction, angina, stroke, diabetes mellitus, renal disease,renal failure, systemic sclerosis, intrauterine growth restriction(IUGR), and fetal growth restriction.

In another embodiment, the angiotensinogen-associated disease isselected from the group consisting of hypertension, hypertensive heartdisease, hypertensive nephropathy, pregnancy-associated hypertension,atherosclerosis, arteriosclerosis, chronic kidney disease,glomerulosclerosis, coarctation of the aorta, aortic aneurism,ventricular fibrosis, Cushing's syndrome, and other glucocorticoidexcess states including chronic steroid therapy, pheochromocytoma,primary aldosteronism and other mineralocorticoid excess states, sleepapnea, thyroid/parathyroid disease, heart failure, myocardialinfarction, stroke, diabetes mellitus, renal failure, and systemicsclerosis.

In one embodiment, the angiotensinogen-associated disease ispregnancy-associated hypertension (e.g., pregnancy-induced hypertension,preeclampsia, and eclampsia) and administration of an iRNA of theinvention to a subject results in a decrease in maternal blood pressure;a decrease in maternal albuminuria; an increase in uteroplacental unitweight; an increase in fetal weight; normalization of the fetalbrain:liver ratio; a decrease in AGT mRNA expression in the maternalliver and no significant decrease in hAGT mRNA expression in theplacenta; an increase in overall placenta size; an increase in the sizeof the villous placenta; no significant change in the size of thetrophospongium of the placenta; a reduction in the ratio of sFLT1:PLGFmRNA expression in the maternal kidney; a reduction in the ratio ofserum sFLT1:PLGF levels; and/or a decrease in the level of agonisticautoantibodies to AT1.

In one embodiment, the double-stranded RNAi agent is administered at adose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about50 mg/kg. In a preferred embodiment, the double-stranded RNAi agent isadministered at a dose of about 10 mg/kg, about 30 mg/kg, or about 3.0mg/kg. In one embodiment, the double-stranded RNAi agent is administeredat a dose of about 10 mg/kg. In one embodiment, the double-stranded RNAiagent is administered at a dose of about 0.5 mg/kg twice per week. Inanother embodiment, the double-stranded RNAi agent is administered at adose of about 10 mg/kg every other week. In another embodiment, thedouble-stranded RNAi agent is administered at a dose of about 0.5-1.0mg/kg once per week. In another embodiment, the RNAi agent isadministered about once per week, once per month, once every other twomonths, or once a quarter (i.e., once every three months) at a dose ofabout 0.1 mg/kg to about 5.0 mg/kg.

In one embodiment, the double-stranded RNAi agent is administeredsubcutaneously or intravenously.

In one embodiment, the RNAi agent is administered in two or more doses.

In one embodiment, the RNAi agent is administered at intervals selectedfrom the group consisting of once every about 12 hours, once every about24 hours, once every about 48 hours, once every about 72 hours, and onceevery about 96 hours.

In one embodiment, the RNAi agent is administered twice per week.

In one embodiment, the RNAi agent is administered every other week.

In certain embodiments, the RNAi agent is administered once per month.

In certain embodiments, the RNAi agent is administered once every othermonth.

In certain embodiments, the RNAi agent is administered once per quarter(i.e., every three months).

In yet another embodiment, the methods further comprise administering tothe subject, an additional therapeutic agent. In some embodiments, theadditional therapeutic agent is selected from the group consisting of adiuretic, an angiotensin converting enzyme (ACE) inhibitor, anangiotensin II receptor antagonist, a beta-blocker, a vasodialator, acalcium channel blocker, an aldosterone antagonist, an alpha₂-agonist, arenin inhibitor, an alpha-blocker, a peripheral acting adrenergic agent,a selective D1 receptor partial agonist, a nonselective alpha-adrenergicantagonist, a synthetic, steroidal antimineralocorticoid agent, or acombination of any of the foregoing, and a hypertension therapeuticagent formulated as a combination of agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the renin-angiotensin-aldosterone system (RAAS)including an indication of the various points in the system which havebeen the targets for therapeutic intervention (from Zaman, et al. (2002)Nat Rev Drug Disc 1:621).

FIG. 2A is a graph depicting the reduction in mean arterial bloodpressure in pregnant transgenic rats following administration ofAD-60771.

FIG. 2B is a graph depicting reduction of serum albumin in pregnanttransgenic rats following administration of AD-60771.

FIG. 3A is a graph depicting increased uteroplacental unit weightfollowing maternal administration of AD-60771 demonstrating theimprovement of fetal outcome following maternal administration ofAD-60771.

FIG. 3B is a graph depicting increased fetal weight following maternaladministration of AD-60771 demonstrating the improvement of fetaloutcome following maternal administration of AD-60771.

FIG. 3C is a graph depicting a normalized fetal brain:liver ratiofollowing maternal administration of AD-60771 demonstrating theimprovement of fetal outcome following maternal administration ofAD-60771.

FIG. 4A is a graph depicting reduction of hAGT mRNA in the maternalliver following administration of AD-60771 demonstrating that the iRNAdoes not enter the placental barrier.

FIG. 4B is a graph depicting that there is no significant reduction ofhAGT mRNA in the placenta demonstrating that the iRNA does not enter theplacental barrier.

FIG. 5 is a graph depicting the tissue exposure of the maternal liver,placenta and fetal liver to AD-60771.

FIG. 6A is a placental section from a pregnant wild-type ratimmunohistochemically stained for cytokeratin.

FIG. 6B is a placental section from an untreated pregnant PE ratimmunohistochemically stained for cytokeratin.

FIG. 6C is a placental section from a pregnant PE rat administeredAD-60771 immunohistochemically stained for cytokeratin.

FIG. 6D is a graph depicting the size of the mesometrial triangle of apregnant PE rat administered AD-60771 and an untreated pregnant PE rat.

FIG. 6E is a graph depicting the size of the trophospongium of apregnant PE rat administered AD-60771 and an untreated pregnant PE rat.

FIG. 6F is a graph depicting the size of the placenta of a pregnant PErat administered AD-60771 and an untreated pregnant PE rat.

FIG. 6G is a graph depicting the size of the labyrinth of a pregnant PErats administered AD-60771 and an untreated pregnant PE rat.

FIG. 7A is a graph depicting a reduction in the amount of mRNA of theanti-angiogenic factor sFLT1 in the maternal kidney followingadministration of AD-60771.

FIG. 7B is a graph depicting a reduction in the amount of mRNA of theangiogenic factor PLGF in the maternal kidney following administrationof AD-60771.

FIG. 7C is a graph depicting a reduction in the amount of mRNA of theanti-angiogenic factor sFLT1 in the placenta following maternaladministration of AD-60771.

FIG. 7D is a graph depicting a reduction in the amount of mRNA of theangiogenic factor PLGF in the placenta following maternal administrationof AD-60771.

FIG. 8 is a graph depicting the reduction in AT1-AA levels in PE ratsadministered AD-60771 as assessed by the impact of AT1-AA isolated fromcontrol PE rats and pregnant PE rats on the spontaneous beating rate ofneonatal rat cardiomyocytes.

FIG. 9A is a graph depicting the reduction in serum Angiotensin II (Ang2-10) levels in pregnant PE rats administered AD-60771 as compared tonon-pregnant PE rats and as compared to pregnant control Sprague-Dawleyrats.

FIG. 9B is a graph depicting the reduction in serum human AGT (hAGT) andrat AGT (rAGT) levels in pregnant PE rats administered AD-60771 ascompared to non-pregnant PE rats and as compared to pregnant controlSprague-Dawley rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an angiotensinogen (AGT) gene. The gene may be within acell, e.g., a cell within a subject, such as a human.

The present invention also provides methods for treating a subjecthaving a disorder that would benefit from inhibiting or reducing theexpression of an AGT gene, e.g., an angiotensinogen-associated disease,such as hypertension or pregnancy-associated hypertension, using iRNAcompositions which effect the RNA-induced silencing complex(RISC)-mediated cleavage of RNA transcripts of an AGT gene.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofan AGT gene. In certain embodiments, the iRNAs of the invention includean RNA strand (the antisense strand) which can include longer lengths,for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60,22-43, 27-53 nucleotides in length with a region of at least 19contiguous nucleotides that is substantially complementary to at least apart of an mRNA transcript of an AGT gene. These iRNAs with the longerlength antisense strands include a second RNA strand (the sense strand)of 20-60 nucleotides in length wherein the sense and antisense strandsform a duplex of 18-30 contiguous nucleotides. The use of these iRNAsenables the targeted degradation of mRNAs of the corresponding gene (AGTgene) in mammals. Very low dosages of the iRNAs of the invention, inparticular, can specifically and efficiently mediate RNA interference(RNAi), resulting in significant inhibition of expression of thecorresponding gene (AGT gene). Using in vitro and in vivo assays, thepresent inventors have demonstrated that iRNAs targeting anangiotensinogen gene can mediate RNAi, resulting in significantinhibition of expression of AGT, as well as reducing the symptomsassociated with an angiotensinogen-associated disease, such aspregnancy-associated hypertension (e.g., pregnancy-induced hypertension,preeclampsia, and eclampsia). Thus, methods and compositions includingthese iRNAs are useful for treating a subject having anangiotensinogen-associated disease, such as hypertension.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of anangiotensinogen gene as well as compositions, uses, and methods fortreating subjects having diseases and disorders that would benefit frominhibition and/or reduction of the expression of AGT.

Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges oftolerances in the art. As used herein, “angiotensinogen,” usedinterchangeably with the term “AGT” refers to the well-known gene andpolypeptide, also known in the art as Serpin Peptidase Inhibitor, CladeA, Member 8; Alpha-1 Antiproteinase; Antitrypsin; SERPINA8; AngiotensinI; Serpin A8; Angiotensin II; Alpha-1 Antiproteinase angiotensinogen;antitrypsin; pre-angiotensinogen2; ANHU; Serine Proteinase Inhibitor;and Cysteine Proteinase Inhibitor.

The term “AGT” includes human AGT, the amino acid and complete codingsequence of which may be found in for example, GenBank Accession No.GI:188595658 (NM_000029.3; SEQ ID NO:1); Macaca fascicularis AGT, theamino acid and complete coding sequence of which may be found in forexample, GenBank Accession No. GI: 90075391 (AB170313.1: SEQ ID NO:3);mouse (Mus musculus) AGT, the amino acid and complete coding sequence ofwhich may be found in for example, GenBank Accession No. GI: 113461997(NM_007428.3; SEQ ID NO:5); and rat AGT (Rattus norvegicus) AGT theamino acid and complete coding sequence of which may be found in forexample, for example GenBank Accession No. GI:51036672 (NM_134432; SEQID NO:7).

Additional examples of AGT mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, OMIM, and theMacaca genome project web site.

The term“AGT,” as used herein, also refers to naturally occurring DNAsequence variations of the AGT gene, such as a single nucleotidepolymorphism (SNP) in the AGT gene. Exemplary SNPs may be found in thedbSNP database available atwww.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=183. Non-limitingexamples of sequence variations within the AGT gene include, forexample, those described in U.S. Pat. No. 5,589,584, the entire contentsof which are incorporated herein by reference. For example, sequencevariations within the AGT gene may include as a C→T at position −532(relative to the transcription start site); a G→A at position −386; aG→A at position −218; a C→T at position −18; a G→A and a A→C at position−6 and −10; a C→T at position +10 (untanslated); a C→T at position +521(T174M); a T→C at position +597 (P199P); a T→C at position +704 (M235T;also see, e.g., Reference SNP (refSNP) Cluster Report: rs699, availableat www.ncbi.nlm.nih.gov/SNP); a A→G at position +743 (Y248C); a C→T atposition +813 (N271N); a G→A at position +1017 (L339L); a C→A atposition +1075 (L359M); and/or a G→A at position +1162 (V388M).

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an AGT gene, including mRNA that is a product of RNA processing of aprimary transcription product. In one embodiment, the target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of an AGTgene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of AGT in a cell, e.g., a cell within a subject, such asa mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., an AGTtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double-strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., an AGT gene. Accordingly, the term“siRNA” is also used herein to refer to an RNAi as described above.

In certain embodiments, the RNAi agent may be a single-stranded siRNA(ssRNAi) that is introduced into a cell or organism to inhibit a targetmRNA. Single-stranded RNAi agents bind to the RISC endonuclease,Argonaute 2, which then cleaves the target mRNA. The single-strandedsiRNAs are generally 15-30 nucleotides and are chemically modified. Thedesign and testing of single-stranded siRNAs are described in U.S. Pat.No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double-stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., an AGT gene. In some embodiments ofthe invention, a double-stranded RNA (dsRNA) triggers the degradation ofa target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/or amodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The majority of nucleotides of each strand of a dsRNA molecule may beribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/ormodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In certain embodiments, an RNAi agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., an AGT gene, without wishing to be bound bytheory, long double-stranded RNA introduced into cells is broken downinto siRNA by a Type III endonuclease known as Dicer (Sharp et al.(2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme,processes the dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). The siRNAs are then incorporated into an RNA-induced silencingcomplex (RISC) where one or more helicases unwind the siRNA duplex,enabling the complementary antisense strand to guide target recognition(Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriatetarget mRNA, one or more endonucleases within the RISC cleave the targetto induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., an AGTtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double-stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Incertain embodiments, the overhang on the sense strand or the antisensestrand, or both, can include extended lengths longer than 10nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30nucleotides, or 10-15 nucleotides in length. In certain embodiments, anextended overhang is on the sense strand of the duplex. In certainembodiments, an extended overhang is present on the 3′end of the sensestrand of the duplex. In certain embodiments, an extended overhang ispresent on the 5′end of the sense strand of the duplex. In certainembodiments, an extended overhang is on the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the3′end of the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 5′end of the antisense strand of theduplex. In certain embodiments, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double-stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” RNAi agent is a dsRNA that is double-strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withnucleotide overhangs at one end (i.e., agents with one overhang and oneblunt end) or with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a AGT mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an AGT nucleotide sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches can be in the internal orterminal regions of the molecule. Generally, the most toleratedmismatches are in the terminal regions, e.g., within 5, 4, 3, or 2nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12,and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding AGT). For example, a polynucleotide iscomplementary to at least a part of an AGT mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding AGT.

Accordingly, in some embodiments, the antisense strand polynucleotidesdisclosed herein are fully complementary to the target AGT sequence. Inother embodiments, the antisense strand polynucleotides disclosed hereinare substantially complementary to the target AGT sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1, suchas about 85%, about 90%, or about 95% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target AGTsequence, and wherein the sense strand polynucleotide comprises acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to the equivalent region of the nucleotidesequence of SEQ ID NO:2, or a fragment of any one of SEQ ID NO:2, suchas about 85%, about 90%, or about 95% complementary.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

In one aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisense RNAmolecule that inhibits a target mRNA via an antisense inhibitionmechanism. The single-stranded antisense RNA molecule is complementaryto a sequence within the target mRNA. The single-stranded antisenseoligonucleotides can inhibit translation in a stoichiometric manner bybase pairing to the mRNA and physically obstructing the translationmachinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Thesingle-stranded antisense RNA molecule may be about 15 to about 30nucleotides in length and have a sequence that is complementary to atarget sequence. For example, the single-stranded antisense RNA moleculemay comprise a sequence that is at least about 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from any one of the antisense sequencesdescribed herein.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an AGT,” as used herein, includesinhibition of expression of any AGT gene (such as, e.g., a mouse AGTgene, a rat AGT gene, a monkey AGT gene, or a human AGT gene) as well asvariants or mutants of an AGT gene that encode an AGT protein.

“Inhibiting expression of an AGT gene” includes any level of inhibitionof an AGT gene, e.g., at least partial suppression of the expression ofan AGT gene, such as an inhibition by at least about 20%. In certainembodiments, inhibition is by at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%.

The expression of an AGT gene may be assessed based on the level of anyvariable associated with AGT gene expression, e.g., AGT mRNA level orAGT protein level. Inhibition may be assessed by a decrease in anabsolute or relative level of one or more of these variables comparedwith a control level. The control level may be any type of control levelthat is utilized in the art, e.g., a pre-dose baseline level, or a leveldetermined from a similar subject, cell, or sample that is untreated ortreated with a control (such as, e.g., buffer only control or inactiveagent control).

In one embodiment, at least partial suppression of the expression of anAGT gene, is assessed by a reduction of the amount of AGT mRNA which canbe isolated from or detected in a first cell or group of cells in whichan AGT gene is transcribed and which has or have been treated such thatthe expression of an AGT gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing”or “delivering the iRNA into the cell” by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an iRNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an iRNA into a cell may be invitro and/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below and/or areknown in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in AGT expression; a human at risk for a disease,disorder or condition that would benefit from reduction in AGTexpression; a human having a disease, disorder or condition that wouldbenefit from reduction in AGT expression; and/or human being treated fora disease, disorder or condition that would benefit from reduction inAGT expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with unwanted AGTexpression, e.g., angiotensin II type 1 receptor activation (AT₁R)(e.g., hypertension, chronic kidney disease, stroke, myocardialinfarction, heart failure, aneurysms, peripheral artery disease, heartdisease, increased oxidative stress, e.g., increased superoxideformation, inflammation, vasoconstriction, sodium and water retention,potassium and magnesium loss, renin suppression, myocyte and smoothmuscle hypertrophy, increased collagen sysnthesis, stimulation ofvascular, myocardial and renal fibrosis, increased rate and force ofcardiac contractions, altered heart rate, e.g., increased arrhythmia,stimulation of plasminogen activator inhibitor 1 (PAI1), activation ofthe sympathetic nervous system, and increased endothelin secretion),symptoms of pregnancy-associated hypertension (e.g., preeclampsia, andeclampsia), including, but not limited to intrauterine growthrestriction (IUGR) or fetal growth restriction, symptoms associated withmalignant hypertension, symptoms associated with hyperaldosteronism;diminishing the extent of unwanted AT₁R activation; stabilization (i.e.,not worsening) of the state of chronic AT₁R activation; amelioration orpalliation of unwanted AT₁R activation (e.g., hypertension, chronickidney disease, stroke, myocardial infarction, heart failure, aneurysms,peripheral artery disease, heart disease, increased oxidative stress,e.g., increased superoxide formation, inflammation, vasoconstriction,sodium and water retention, potassium and magnesium loss, reninsuppression, myocyte and smooth muscle hypertrophy, increased collagensysnthesis, stimulation of vascular, myocardial and renal fibrosis,increased rate and force of cardiac contractions, altered heart rate,e.g., increased arrhythmia, stimulation of plasminogen activatorinhibitor 1 (PAI1), activation of the sympathetic nervous system, andincreased endothelin secretion) whether detectable or undetectable.“Treatment” can also mean prolonging survival as compared to expectedsurvival in the absence of treatment.

The term “lower” in the context of the level of AGT in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more and is preferably down to a level accepted aswithin the range of normal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an AGT gene, refers to a reduction in thelikelihood that a subject will develop a symptom associated with such adisease, disorder, or condition, e.g., a symptom of unwanted AT₁Ractivation, such as a hypertension, chronic kidney disease, stroke,myocardial infarction, heart failure, aneurysms, peripheral arterydisease, heart disease, increased oxidative stress, e.g., increasedsuperoxide formation, inflammation, vasoconstriction, sodium and waterretention, potassium and magnesium loss, renin suppression, myocyte andsmooth muscle hypertrophy, increased collagen sysnthesis, stimulation ofvascular, myocardial and renal fibrosis, increased rate and force ofcardiac contractions, altered heart rate, e.g., increased arrhythmia,stimulation of plasminogen activator inhibitor 1 (PAI1), activation ofthe sympathetic nervous system, and increased endothelin secretion. Thelikelihood of developing, e.g., hypertension, is reduced, for example,when an individual having one or more risk factors for a hypertensioneither fails to develop hypertension or develops hypertension with lessseverity relative to a population having the same risk factors and notreceiving treatment as described herein. The failure to develop adisease, disorder or condition, or the reduction in the development of asymptom associated with such a disease, disorder or condition (e.g., byat least about 10% on a clinically accepted scale for that disease ordisorder), or the exhibition of delayed symptoms delayed (e.g., by days,weeks, months or years) is considered effective prevention.

As used herein, the term “angiotensinogen-associated disease” or“AGT-associated disease,” is a disease or disorder that is caused by, orassociated with renin-angiotensin-aldosterone system (RAAS) activation,or a disease or disorder the symptoms of which or progression of whichresponds to RAAS inactivation. The term “angiotensinogen-associateddisease” includes a disease, disorder or condition that would benefitfrom reduction in AGT expression. Such diseases are typically associatedwith high blood pressure. Non-limiting examples ofangiotensinogen-associated diseases include hypertension, e.g.,borderline hypertension (also known as prehypertension), primaryhypertension (also known as essential hypertension or idiopathichypertension), secondary hypertension (also known as inessentialhypertension), hypertensive emergency (also known as malignanthypertension), hypertensive urgency, isolated systolic or diastolichypertension, pregnancy-associated hypertension (e.g., preeclampsia,eclampsia, and post-partum preelampsia), diabetic hypertension,resistant hypertension, refractory hypertension, paroxysmalhypertension, renovascular hypertension (also known as renalhypertension), Goldblatt hypertension, ocular hypertension, glaucoma,pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy (including peripheral vascular disease),diabetic nephropathy, diabetic retinopathy, chronic heart failure,cardiomyopathy, diabetic cardiac myopathy, glomerulosclerosis,coarctation of the aorta, aortic aneurism, ventricular fibrosis,Cushing's syndrome, and other glucocorticoid excess states includingchronic steroid therapy, pheochromocytoma, reninoma, secondaryaldosteronism and other mineralocorticoid excess states, sleep apnea,thyroid/parathyroid disease, heart failure (e.g., left ventricularsystolic dysfunction), myocardial infarction, angina, stroke, diabetesmellitus (e.g., diabetic nephropathy), renal disease e.g., chronickidney disease or diabetic nephropathy optionally in the context ofpregnancy, renal failure, e.g., chronic renal failure, cognitivedysfunction (such as Alzheimer's), and systemic sclerosis (e.g.,scleroderma renal crisis). In certain embodiments, AGT-associateddisease includes intrauterine growth restriction (IUGR) or fetal growthrestriction.

Based on the average of seated blood pressure readings that are properlymeasured during two or more office visits, a subject having a normalblood pressure is one having a systolic pressure of about 90-119 mmHg(about 12-15.9 kPa (kN/m²)) and a diastolic pressure of about 60-79 mmHg(about 8.0-10.5 kPa (kN/m²)); a subject having prehypertension is onehaving a systolic pressure of about 120-139 mmHg (about 16.1-18.5 kPa(kN/m²)) and a diastolic pressure of about 60-79 mmHg (about 8.0-10.5kPa (kN/m²)); a subject having hypertension (e.g., Stage I hypertension)is one having a systolic pressure of about 140-159 mmHg (about 18.7-21.2kPa (kN/m²)) and a diastolic pressure of about 90-99 mmHg (about12.0-13.2 kPa (kN/m²)); and a subject having hypertension (e.g., StageII hypertension) is one having a systolic pressure of about ≥160 mmHg(about ≥21.3 kPa (kN/m²)) and a diastolic pressure of about ≥100 mmHg(about ≥13.3 kPa (kN/m²)). Subjects with blood pressures over 130/80mmHg along with Type 1 or Type 2 diabetes, or kidney disease areconsidered as having hypertension.

In one embodiment, an angiotensinogen-associated disease is primaryhypertension. “Primary hypertension” is a result of environmental orgenetic causes (e.g., a result of no obvious underlying medical cause).

In one embodiment, an angiotensinogen-associated disease is secondaryhypertension. “Secondary hypertension” has an identifiable underlyingdisorder which can be of multiple etiologies, including renal, vascular,and endocrine causes, e.g., renal parenchymal disease (e.g., polycystickidneys, glomerular or interstitial disease), renal vascular disease(e.g., renal artery stenosis, fibromuscular dysplasia), endocrinedisorders (e.g., adrenocorticosteroid or mineralocorticoid excess,pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormoneexcess, hyperparathyroidism), coarctation of the aorta, or oralcontraceptive use.

In one embodiment, an angiotensinogen-associated disease is ahypertensive emergency, e.g., malignant hypertension and acceleratedhypertension. “Accelerated hypertension” is severely elevated bloodpressure (i.e., equal to or greater than a systolic 180 mmHg ordiastolic of 110 mmHg) with direct damage to one or more end organs.Blood pressure must be reduced immediately to prevent further organdamage. “Malignant hypertension” is severely elevated blood pressure(i.e., equal to or greater than a systolic 180 mmHg or diastolic of 110mmHg) with direct damage to one or more end organs and papilledema.Blood pressure must be reduced immediately to prevent further organdamage. Neurologic end-organ damage due to uncontrolled blood pressuremay include hypertensive encephalopathy, cerebral vascularaccident/cerebral infarction; subarachnoid hemorrhage, and/orintracranial hemorrhage. Cardiovascular end-organ damage may includemyocardial ischemia/infarction, acute left ventricular dysfunction,acute pulmonary edema, and/or aortic dissection. Other organ systems mayalso be affected by uncontrolled hypertension, which may lead to acuterenal failure/insufficiency, retinopathy, eclampsia, or microangiopathichemolytic anemia.

In one embodiment, an angiotensinogen-associated disease is ahypertensive urgency. “Hypertensive urgency” is severely elevated bloodpressure (i.e., equal to or greater than a systolic 180 mmHg ordiastolic of 110 mmHg) with no direct damage to one or more organs.Blood pressure can be brought down safely within a few hours.

In one embodiment, an angiotensinogen-associated disease ispregnancy-associated hypertension, e.g., chronic hypertension ofpregnancy, gestational hypertension, preeclampsia, eclampsia,preeclampsia superimposed on chronic hypertension, HELLP syndrome, andgestational hypertension (also known as transient hypertension ofpregnancy, chronic hypertension identified in the latter half ofpregnancy, and pregnancy-induced hypertension (PIH)). A subject having“chronic hypertension of pregnancy” is one having a blood pressureexceeding 140/90 mm Hg before pregnancy or before 20 weeks' gestation.“Gestational hypertension” or “pregnancy-induced hypertension” refers tohypertension with onset in the latter part of pregnancy (>20 weeks'gestation) without any other features of preeclampsia, and followed bynormalization of the blood pressure postpartum. “Mild preeclampsia” isdefined as the presence of hypertension (blood pressure >140/90 mm Hg)on two occasions, at least six hours apart, but without evidence ofend-organ damage, in a woman who was normotensive before 20 weeks'gestation. In a subject with preexisting essential hypertension,preeclampsia is diagnosed if systolic blood pressure has increased by 30mm Hg or if diastolic blood pressure has increased by 15 mm Hg. “Severepreeclampsia” is defined as the presence of 1 of the following symptomsor signs in the presence of preeclampsia; asystolic blood pressure of160 mm Hg or higher or diastolic blood pressure of 110 mm Hg or higheron two occasions at least six hours apart; proteinuria of more than 5 gin a 24-hour collection or more than 3+ on two random urine samplescollected at least four hours apart, pulmonary edema or cyanosis,oliguria (<400 mL in 24 hours), persistent headaches, epigastric painand/or impaired liver function, thrombocytopenia, oligohydramnios,decreased fetal growth, or placental abruption. “Eclampsia” is definedas seizures that cannot be attributable to other causes in a woman withpreeclampsia. “HELLP syndrome” (also known asedema-proteinuria-hypertension gestosis type B) is Hemolysis, ElevatedLiver enzyme levels, and Low Platelet levels in a pregnant subject.

In one embodiment, an angiotensinogen-associated disease is resistanthypertension. “Resistant hypertension” is blood pressure that remainsabove goal (e.g., 140/90 mmHg) in spite of concurrent use of threeantihypertensive agents of different classes, one of which is a thiazidediuretic diuretic. Subjects whose blood pressure is controlled with fouror more medications are also considered to have resistant hypertension.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving an angiotensinogen-associated disease, is sufficient to effecttreatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on the RNAiagent, how the agent is administered, the disease and its severity andthe history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subjecthaving an angiotensinogen-associated disease, is sufficient to preventor ameliorate the disease or one or more symptoms of the disease in asubject susceptible to the disease, i.e., more likely to suffer from thedisease than those in the general population due to one or more factors,e.g., age, weight, pregnancy. Ameliorating the disease includes slowingthe course of the disease or reducing the severity of later-developingdisease. The “prophylactically effective amount” may vary depending onthe iRNA, how the agent is administered, the degree of risk of disease,and the history, age, weight, family history, genetic makeup, the typesof preceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. iRNA employed in the methods of the presentinvention may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to blood or plasma drawn from the subject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of anAGT gene. In one embodiment, the iRNA agent includes double-strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anAGT gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human having an angiotensinogen-associated disease, e.g.,hypertension. The dsRNA includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an AGT gene. The region of complementarityis about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length).Upon contact with a cell expressing the AGT gene, the iRNA inhibits theexpression of the AGT gene (e.g., a human, a primate, a non-primate, ora bird AGT gene) by at least about 10% as assayed by, for example, a PCRor branched DNA (bDNA)-based method, or by a protein-based method, suchas by immunofluorescence analysis, using, for example, western blottingor flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an AGTgene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is about 15 to about 20 nucleotides inlength, about 25 to about 30 nucleotides in length, or about 15 to about23 nucleotides in length. In general, the dsRNA is long enough to serveas a substrate for the Dicer enzyme. For example, it is well-known inthe art that dsRNAs longer than about 21-23 nucleotides in length mayserve as substrates for Dicer. As the ordinarily skilled person willalso recognize, the region of an RNA targeted for cleavage will mostoften be part of a larger RNA molecule, often an mRNA molecule. Whererelevant, a “part” of an mRNA target is a contiguous sequence of an mRNAtarget of sufficient length to allow it to be a substrate forRNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target AGT expression is not generated in the target cell by cleavageof a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof Furthermore, the nucleotide(s) of an overhang can bepresent on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA. As discussed herein, extended overhang of up to30 nucleotides in length are also contemplated in various embodiments ofthe invention.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems®, Inc. iRNA compounds of the invention may be prepared usinga two-step procedure. First, the individual strands of thedouble-stranded RNA molecule are prepared separately. Then, thecomponent strands are annealed. The individual strands of the siRNAcompound can be prepared using solution-phase or solid-phase organicsynthesis or both. Organic synthesis offers the advantage that theoligonucleotide strands comprising unnatural or modified nucleotides canbe easily prepared. Single-stranded oligonucleotides of the inventioncan be prepared using solution-phase or solid-phase organic synthesis orboth.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in any one of Tables 3,4, 7, 8, 11, 13, and 15, and the corresponding antisense strand of thesense strand is selected from the group of sequences of any one ofTables 3, 4, 7, 8, 11, 13, and 15. In this aspect, one of the twosequences is complementary to the other of the two sequences, with oneof the sequences being substantially complementary to a sequence of anmRNA generated in the expression of an AGT gene. As such, in thisaspect, a dsRNA will include two oligonucleotides, where oneoligonucleotide is described as the sense strand in any one of Tables 3,4, 7, 8, 11, 13, and 15, and the second oligonucleotide is described asthe corresponding antisense strand of the sense strand in any one ofTables 3, 4, 7, 8, 11, 13, and 15. In one embodiment, the substantiallycomplementary sequences of the dsRNA are contained on separateoligonucleotides. In another embodiment, the substantially complementarysequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although some of the sequences in Tables 3,4, 7, 8, 11, 13, and 15 are described as modified and/or conjugatedsequences, the RNA of the iRNA of the invention e.g., a dsRNA of theinvention, may comprise any one of the sequences set forth in Tables 3,4, 7, 8, 11, 13, and 15 that is un-modified, un-conjugated, and/ormodified and/or conjugated differently than described therein.

In another aspect, a double-stranded ribonucleic acid (dsRNA) of theinvention for inhibiting expression of angiotensinogen comprises,consists essentially of, or consists of a sense strand and an antisensestrand, wherein the sense strand comprises the nucleotide sequence of asense strand in Table 3, 4, 7, 8, 11, 13, and 15 and the antisensestrand comprises the nucleotide sequence of the corresponding antisensestrand in Tables 3, 4, 7, 8, 11, 13, and 15.

The skilled person is well aware that dsRNAs having a duplex structureof about 20 to 23 base pairs, e.g., 21, base pairs have been hailed asparticularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can also be effective (Chu and Rana (2007)RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 3, 4, 7, 8, 11,13, and 15, dsRNAs described herein can include at least one strand of alength of minimally 21 nucleotides. It can be reasonably expected thatshorter duplexes having one of the sequences of any one of Tables 3, 4,7, 8, 11, 13, and 15 minus only a few nucleotides on one or both endscan be similarly effective as compared to the dsRNAs described above.Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, ormore contiguous nucleotides derived from one of the sequences of any oneof Tables 3, 4, 7, 8, 11, 13, and 15, and differing in their ability toinhibit the expression of a AGT gene by not more than about 5, 10, 15,20, 25, or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated to be within the scope of the present invention.

In addition, the RNAs provided in any one of Tables 3, 4, 7, 8, 11, 13,and 15 identify a site(s) in a AGT transcript that is susceptible toRISC-mediated cleavage. As such, the present invention further featuresiRNAs that target within one of these sites. As used herein, an iRNA issaid to target within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided in any one of Tables 3,4, 7, 8, 11, 13, and 15 coupled to additional nucleotide sequences takenfrom the region contiguous to the selected sequence in a AGT gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 3, 4,7, 8, 11, 13, and 15 represent effective target sequences, it iscontemplated that further optimization of inhibition efficiency can beachieved by progressively “walking the window” one nucleotide upstreamor downstream of the given sequences to identify sequences with equal orbetter inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 3, 4, 7, 8, 11, 13, and 15, further optimization couldbe achieved by systematically either adding or removing nucleotides togenerate longer or shorter sequences and testing those sequencesgenerated by walking a window of the longer or shorter size up or downthe target RNA from that point. Again, coupling this approach togenerating new candidate targets with testing for effectiveness of iRNAsbased on those target sequences in an inhibition assay as known in theart and/or as described herein can lead to further improvements in theefficiency of inhibition. Further still, such optimized sequences can beadjusted by, e.g., the introduction of modified nucleotides as describedherein or as known in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of an AGT gene, generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an AGT gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of an AGTgene is important, especially if the particular region ofcomplementarity in an AGT gene is known to have polymorphic sequencevariation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(m)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

In some embodiments, the oligonucleotide of the invention comprises oneor more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAis unlocked acyclic nucleic acid, wherein any of the bonds of the sugarhas been removed, forming an unlocked “sugar” residue. In one example,UNA also encompasses monomer with bonds between C1′-C4′ have beenremoved (i.e. the covalent carbon-oxygen-carbon bond between the C1′ andC4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH2)-S-2; 4′-(CH₂)₂—O-2′(ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or“cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see, e.g., U.S.Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g.,U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; seee.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., U.S.Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H,C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No.7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J.Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogsthereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents ofeach of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an RNAi agent.Suitable phosphate mimics are disclosed in, for example US PatentPublication No. 2012/0157511, the entire contents of which areincorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in U.S. Provisional Application No. 61/561,710, filed onNov. 18, 2011, or in PCT/US2012/065691, filed on Nov. 16, 2012, theentire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710 or PCTApplication No. PCT/US2012/065691, a superior result may be obtained byintroducing one or more motifs of three identical modifications on threeconsecutive nucleotides into a sense strand and/or antisense strand ofan RNAi agent, particularly at or near the cleavage site. In someembodiments, the sense strand and antisense strand of the RNAi agent mayotherwise be completely modified. The introduction of these motifsinterrupts the modification pattern, if present, of the sense and/orantisense strand. The RNAi agent may be optionally conjugated with aGalNAc derivative ligand, for instance on the sense strand. Theresulting RNAi agents present superior gene silencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an RNAi agent, the gene silencingacitivity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double-stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., angiotensinogen(AGT) gene) in vivo. The RNAi agent comprises a sense strand and anantisense strand. Each strand of the RNAi agent may range from 12-30nucleotides in length. For example, each strand may be between 14-30nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides inlength, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides inlength, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplexdouble-stranded RNA (“dsRNA”), also referred to herein as an “RNAiagent.” The duplex region of an RNAi agent may be 12-30 nucleotide pairsin length. For example, the duplex region can be between 14-30nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23nucleotide pairs in length. In another example, the duplex region isselected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. As discussed herein, extended overhang of up to30 nucleotides in length are also contemplated in various embodiments ofthe invention. The first and second strands can also be joined, e.g., byadditional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate intemucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate intemucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In one embodiment, everynucleotide in the sense strand and the antisense strand of the RNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In one embodiment each residue is independentlymodified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif.Optionally, the RNAi agent further comprises a ligand (preferablyGalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3 ‘ terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3’ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when thedouble-stranded nucleic acid is introduced into a mammalian cell; andwherein the sense strand contains at least one motif of three 2′-Fmodifications on three consecutive nucleotides, where at least one ofthe motifs occurs at or near the cleavage site. The antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region region which is at least 25 nucleotides in length, andthe second strand is sufficiently complementary to a target mRNA alongat least 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradajacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two, orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):

5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern. In one embodiment, the YYY motif occurs at or nearthe cleavage site of the sense strand. For example, when the RNAi agenthas a duplex region of 17-23 nucleotides in length, the YYY motif canoccur at or the vicinity of the cleavage site (e.g.: can occur atpositions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12,13) of—the sense strand, the count starting from the 1^(st) nucleotide,from the 5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′n _(p)—N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Ib);

5′n _(p)—N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′  (Ic); or

5′n _(p)—N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides. Each of X, Y and Z may be the same or different from eachother.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′n _(p)—N_(a)—YYY—N_(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n _(q)′—N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′n _(q)′—N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′—N_(a)′-n _(p)′3′  (IIb);

5′n _(q)′—N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n _(p)′3′  (IIc); or

5′n _(q)′—N_(a)′—X′X′X′—N_(a)′-n _(p)′3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:

5′n _(p)′—N_(a)—Y′Y′Y′—N_(a)′-n _(q)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Each of X′, Y′ and Z′ may be thesame or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′

antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′   (III)

wherein:

i, j, k, and 1 are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′n _(p)—N_(a)—Y Y Y—N_(a)-n _(q)3′

3′n _(p)′—N_(a)′—Y′Y′Y′—N_(a) ′n _(q)′5′   (IIIa)

5′n _(p)—N_(a)—Y Y Y—N_(b)—Z Z Z—N_(a)-n _(q)3′

3′n _(p)′—N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a) ′n _(q)′5′   (IIIb)

5′n _(p)—N_(a)—X X X—N_(b)—Y Y Y—N_(a)-n _(q)3′

3′n _(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n _(q)′5′   (IIIc)

5′n _(p)—N_(a)—X X X—N_(b)—Y Y Y—N_(b)—Z Z Z—N_(a)-n _(q)3′

3′n _(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)-n _(q)′5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 3, 4, 7, 8, 11, 13, and 15. These agents may furthercomprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include AGTand above (e.g., AGT, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., AGT, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in thepresent invention include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers In some embodiments, the conjugate or ligand described hereincan be attached to an iRNA oligonucleotide with various linkers that canbe cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox cleavable linking groups In one embodiment, a cleavable linkinggroup is a redox cleavable linking group that is cleaved upon reductionor oxidation. An example of reductively cleavable linking group is adisulphide linking group (—S—S—). To determine if a candidate cleavablelinking group is a suitable “reductively cleavable linking group,” orfor example is suitable for use with a particular iRNA moiety andparticular targeting agent one can look to methods described herein. Forexample, a candidate can be evaluated by incubation with dithiothreitol(DTT), or other reducing agent using reagents know in the art, whichmimic the rate of cleavage which would be observed in a cell, e.g., atarget cell. The candidates can also be evaluated under conditions whichare selected to mimic blood or serum conditions. In one, candidatecompounds are cleaved by at most about 10% in the blood. In otherembodiments, useful candidate compounds are degraded at least about 2,4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in thecell (or under in vitro conditions selected to mimic intracellularconditions) as compared to blood (or under in vitro conditions selectedto mimic extracellular conditions). The rate of cleavage of candidatecompounds can be determined using standard enzyme kinetics assays underconditions chosen to mimic intracellular media and compared toconditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid cleavable linking groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P⁴, P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L⁵C represent a monosaccharide, such        as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having an antgiotensin associated disease or condition) canbe achieved in a number of different ways. For example, delivery may beperformed by contacting a cell with an iRNA of the invention either invitro or in vivo. In vivo delivery may also be performed directly byadministering a composition comprising an iRNA, e.g., a dsRNA, to asubject. Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther 11:267-274)and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006)Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNAinterference has also shown success with local delivery to the CNS bydirect injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMCNeurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528;Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the AGT gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdrl gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an AGT gene. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery, e.g., bysubcutaneous (SC) or intravenous (IV) delivery. Another example iscompositions that are formulated for direct delivery into the brainparenchyma, e.g., by infusion into the brain, such as by continuous pumpinfusion. The pharmaceutical compositions of the invention may beadministered in dosages sufficient to inhibit expression of an AGT gene.In general, a suitable dose of an iRNA of the invention will be in therange of about 0.001 to about 200.0 milligrams per kilogram body weightof the recipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. For example, the dsRNA can be administeredat about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg,about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per singledose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the dsRNA is administered at a dose of about 0.1to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the dsRNA isadministered at a dose of about 10 mg/kg to about 30 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered, e.g., subcutaneously orintravenously, a single therapeutic amount of iRNA, such as about 0.1,0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375,0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65,0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925,0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29,29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In some embodiments, subjects are administered, e.g., subcutaneously orintravenously, multiple doses of a therapeutic amount of iRNA, such as adose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A multi-dose regimine mayinclude administration of a therapeutic amount of iRNA daily, such asfor two days, three days, four days, five days, six days, seven days, orlonger.

In other embodiments, subjects are administered, e.g., subcutaneously orintravenously, a repeat dose of a therapeutic amount of iRNA, such as adose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A repeat-dose regiminemay include administration of a therapeutic amount of iRNA on a regularbasis, such as every other day, every third day, every fourth day, twicea week, once a week, every other week, or once a month. In certainembodiments, the iRNA is administered about once per month to about onceper quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administeredon a less frequent basis.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered by subcutaneousadministration.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose. A higher dose may be administered initially(i.e., a loading dose), followed by a lower dosage for a sustainedperiod of time.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.In certain embodiments, the iRNA is administered about once per month toabout once per quarter (i.e., about once every three months).

The pharmaceutical composition can be administered for an indefiniteperiod of time, e.g., in a subject experiencing elevated blood pressuredue to obesity, or during the time at which the cause of an elevatedlevel of AGT is present, e.g., during pregnancy induced high bloodpressure.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate iRNA. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. USA., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside Gm′ or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNA agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA agentcan be delivered, for example, subcutaneously by infection in order todeliver iRNA agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMAdioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMGtetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG- DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 Cl2-200/D SPC/Chol/PEG-D SG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed April 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx,

heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx,—C(═O)NRxRy, —SOnRx and —SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1L), was added a solution of 514 (10 g,0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere.After complete addition, reaction mixture was warmed to room temperatureand then heated to reflux for 4 h. Progress of the reaction wasmonitored by TLC. After completion of reaction (by TLC) the mixture wascooled to 0° C. and quenched with careful addition of saturated Na2SO4solution. Reaction mixture was stirred for 4 h at room temperature andfiltered off. Residue was washed well with THF. The filtrate andwashings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCland stirred for 20 minutes at room temperature. The volatilities werestripped off under vacuum to furnish the hydrochloride salt of 515 as awhite solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H),5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]−232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2. 74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR 8=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, LV., Popovich NG., and Ansel HC., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich NG., and AnselHC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich NG., and AnselHC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen,LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen™; Carlsbad, Calif.),Lipofectamine2000™ (Invitrogen™; Carlsbad, Calif.), 293fectin™(Invitrogen™; Carlsbad, Calif.), Cellfectin™ (Invitrogen™; Carlsbad,Calif.), DMRIE-C™ (Invitrogen™; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen™; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen™;Carlsbad, Calif.), Lipofectamine™ (Invitrogen™; Carlsbad, Calif.),iRNAMAX (Invitrogen™; Carlsbad, Calif.), Oligofectamine™ (Invitrogen™;Carlsbad, Calif.), Optifect™ (Invitrogen™; Carlsbad, Calif.),X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse,Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse,Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse,Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam®Reagent (Promega®; Madison, Wis.), TransFast™ Transfection Reagent(Promega®; Madison, Wis.), Tfx™-20 Reagent (Promega®; Madison, Wis.),Tfx™-50 Reagent (Promega®; Madison, Wis.), DreamFect™ (OZ Biosciences;Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France),TransPassa D1 Transfection Reagent (New England Biolabs™; Ipswich,Mass., USA), LyoVec™/LipoGen™ (Invitrogen™; San Diego, Calif., USA),PerFectin Transfection Reagent (Genlantis™; San Diego, Calif., USA),NeuroPORTER® Transfection Reagent (Genlantis™; San Diego, Calif., USA),GenePORTER® Transfection reagent (Genlantis™; San Diego, Calif., USA),GenePORTER® 2 Transfection reagent (Genlantis™; San Diego, Calif., USA),Cytofectin Transfection Reagent (Genlantis™; San Diego, Calif., USA),BaculoPORTER® Transfection Reagent (Genlantis™; San Diego, Calif., USA),TroganPORTER™ transfection Reagent (Genlantis™; San Diego, Calif., USA),RiboFect (Bioline™; Taunton, Mass., USA), PlasFect (Bioline™; Taunton,Mass., USA), UniFECTOR (B-Bridge International™; Mountain View, Calif.,USA), SureFECTOR (B-Bridge International™; Mountain View, Calif., USA),or HiFect™ (B-Bridge International™, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-iRNA mechanism and which are useful intreating a hemolytic disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby AGT expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VII. Methods of the Invention

The present invention provides therapeutic and prophylactic methodswhich include administering to a subject having, or prone to developing,an AGT-associated disease, disorder, and/or condition (e.g.,hypertension), pharmaceutical compositions comprising an iRNA agent, orvector comprising an iRNA of the invention.

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in AGTexpression, e.g., a AGT-associated disease, e.g. hypertension, e.g.,borderline hypertension (also known as prehypertension), primaryhypertension (also known as essential hypertension or idiopathichypertension), secondary hypertension (also known as inessentialhypertension), hypertensive emergency (also known as malignanthypertension), hypertensive urgency, isolated systolic or diastolichypertension, pregnancy-associated hypertension (e.g., preeclampsia,eclampsia, and post-partum preelampsia), diabetic hypertension,resistant hypertension, refractory hypertension, paroxysmalhypertension, renovascular hypertension (also known as renalhypertension), Goldblatt hypertension, ocular hypertension, glaucoma,pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy (including peripheral vascular disease),diabetic nephropathy, diabetic retinopathy, chronic heart failure,cardiomyopathy, diabetic cardiac myopathy, glomerulosclerosis,coarctation of the aorta, aortic aneurism, ventricular fibrosis,Cushing's syndrome, and other glucocorticoid excess states includingchronic steroid therapy, pheochromocytoma, reninoma, secondaryaldosteronism and other mineralocorticoid excess states, sleep apnea,thyroid/parathyroid disease, heart failure (e.g., left ventricularsystolic dysfunction), myocardial infarction, angina, stroke, diabetesmellitus (e.g., diabetic nephropathy), renal disease e.g., chronickidney disease or diabetic nephropathy optionally in the context ofpregnancy, renal failure, e.g., chronic renal failure, cognitivedysfunction (such as Alzheimer's), and systemic sclerosis (e.g.,scleroderma renal crisis). In certain embodiments, AGT-associateddisease includes intrauterine growth restriction (IUGR) or fetal growthrestriction. The treatment methods (and uses) of the invention includeadministering to the subject, e.g., a human, a therapeutically effectiveamount of an iRNA agent targeting an AGT gene or a pharmaceuticalcomposition comprising an iRNA agent targeting an AGT gene, therebytreating the subject having a disorder that would benefit from reductionin AGT expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin AGT expression, e.g., a AGT-associated disease, e.g., hypertension,e.g., borderline hypertension (also known as prehypertension), primaryhypertension (also known as essential hypertension or idiopathichypertension), secondary hypertension (also known as inessentialhypertension), hypertensive emergency (also known as malignanthypertension), hypertensive urgency, isolated systolic or diastolichypertension, pregnancy-associated hypertension (e.g., preeclampsia,eclampsia, and post-partum preelampsia), diabetic hypertension,resistant hypertension, refractory hypertension, paroxysmalhypertension, renovascular hypertension (also known as renalhypertension), Goldblatt hypertension, ocular hypertension, glaucoma,pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy (including peripheral vascular disease),diabetic nephropathy, diabetic retinopathy, chronic heart failure,cardiomyopathy, diabetic cardiac myopathy, glomerulosclerosis,coarctation of the aorta, aortic aneurism, ventricular fibrosis,Cushing's syndrome, and other glucocorticoid excess states includingchronic steroid therapy, pheochromocytoma, reninoma, secondaryaldosteronism and other mineralocorticoid excess states, sleep apnea,thyroid/parathyroid disease, heart failure (e.g., left ventricularsystolic dysfunction), myocardial infarction, angina, stroke, diabetesmellitus (e.g., diabetic nephropathy), renal disease e.g., chronickidney disease or diabetic nephropathy optionally in the context ofpregnancy, renal failure, e.g., chronic renal failure, cognitivedysfunction (such as Alzheimer's), and systemic sclerosis (e.g.,scleroderma renal crisis). In certain embodiments, AGT-associateddisease includes intrauterine growth restriction (IUGR) or fetal growthrestriction The methods include administering to the subject atherapeutically effective amount of the iRNA agent, e.g., dsRNA, orvector of the invention, thereby preventing at least one symptom in thesubject having a disorder that would benefit from reduction in AGTexpression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of AGT expression.

In a further aspect, the present invention provides uses of an iRNAagent, e.g., a dsRNA, of the invention targeting an AGT gene orpharmaceutical composition comprising an iRNA agent targeting an AGTgene in the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of AGTexpression, such as a subject having a disorder that would benefit fromreduction in AGT expression, e.g., a AGT-associated disease, e.g.,hypertension, e.g., borderline hypertension (also known asprehypertension), primary hypertension (also known as essentialhypertension or idiopathic hypertension), secondary hypertension (alsoknown as inessential hypertension), hypertensive emergency (also knownas malignant hypertension), hypertensive urgency, isolated systolic ordiastolic hypertension, pregnancy-associated hypertension (e.g.,preeclampsia, eclampsia, and post-partum preelampsia), diabetichypertension, resistant hypertension, refractory hypertension,paroxysmal hypertension, renovascular hypertension (also known as renalhypertension), Goldblatt hypertension, ocular hypertension, glaucoma,pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy (including peripheral vascular disease),diabetic nephropathy, diabetic retinopathy, chronic heart failure,cardiomyopathy, diabetic cardiac myopathy, glomerulosclerosis,coarctation of the aorta, aortic aneurism, ventricular fibrosis,Cushing's syndrome, and other glucocorticoid excess states includingchronic steroid therapy, pheochromocytoma, reninoma, secondaryaldosteronism and other mineralocorticoid excess states, sleep apnea,thyroid/parathyroid disease, heart failure (e.g., left ventricularsystolic dysfunction), myocardial infarction, angina, stroke, diabetesmellitus (e.g., diabetic nephropathy), renal disease e.g., chronickidney disease or diabetic nephropathy optionally in the context ofpregnancy, renal failure, e.g., chronic renal failure, cognitivedysfunction (such as Alzheimer's), and systemic sclerosis (e.g.,scleroderma renal crisis). In certain embodiments, AGT-associateddisease includes intrauterine growth restriction (IUGR) or fetal growthrestriction.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of AGT expression, such as a AGT-associated disease, e.g.,hypertension, e.g., borderline hypertension (also known asprehypertension), primary hypertension (also known as essentialhypertension or idiopathic hypertension), secondary hypertension (alsoknown as inessential hypertension), hypertensive emergency (also knownas malignant hypertension), hypertensive urgency, isolated systolic ordiastolic hypertension, pregnancy-associated hypertension (e.g.,preeclampsia, eclampsia, and post-partum preelampsia), diabetichypertension, resistant hypertension, refractory hypertension,paroxysmal hypertension, renovascular hypertension (also known as renalhypertension), Goldblatt hypertension, ocular hypertension, glaucoma,pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy (including peripheral vascular disease),diabetic nephropathy, diabetic retinopathy, chronic heart failure,cardiomyopathy, diabetic cardiac myopathy, glomerulosclerosis,coarctation of the aorta, aortic aneurism, ventricular fibrosis,Cushing's syndrome, and other glucocorticoid excess states includingchronic steroid therapy, pheochromocytoma, reninoma, secondaryaldosteronism and other mineralocorticoid excess states, sleep apnea,thyroid/parathyroid disease, heart failure (e.g., left ventricularsystolic dysfunction), myocardial infarction, angina, stroke, diabetesmellitus (e.g., diabetic nephropathy), renal disease e.g., chronickidney disease or diabetic nephropathy optionally in the context ofpregnancy, renal failure, e.g., chronic renal failure, cognitivedysfunction (such as Alzheimer's), and systemic sclerosis (e.g.,scleroderma renal crisis). In certain embodiments, AGT-associateddisease includes intrauterine growth restriction (IUGR) or fetal growthrestriction.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of AGT expression, such as aAGT-associated disease, e.g., hypertension, e.g., borderlinehypertension (also known as prehypertension), primary hypertension (alsoknown as essential hypertension or idiopathic hypertension), secondaryhypertension (also known as inessential hypertension), hypertensiveemergency (also known as malignant hypertension), hypertensive urgency,isolated systolic or diastolic hypertension, pregnancy-associatedhypertension (e.g., preeclampsia, eclampsia, and post-partumpreelampsia), diabetic hypertension, resistant hypertension, refractoryhypertension, paroxysmal hypertension, renovascular hypertension (alsoknown as renal hypertension), Goldblatt hypertension, ocularhypertension, glaucoma, pulmonary hypertension, portal hypertension,systemic venous hypertension, systolic hypertension, labilehypertension; hypertensive heart disease, hypertensive nephropathy,atherosclerosis, arteriosclerosis, vasculopathy (including peripheralvascular disease), diabetic nephropathy, diabetic retinopathy, chronicheart failure, cardiomyopathy, diabetic cardiac myopathy,glomerulosclerosis, coarctation of the aorta, aortic aneurism,ventricular fibrosis, Cushing's syndrome, and other glucocorticoidexcess states including chronic steroid therapy, pheochromocytoma,reninoma, secondary aldosteronism and other mineralocorticoid excessstates, sleep apnea, thyroid/parathyroid disease, heart failure (e.g.,left ventricular systolic dysfunction), myocardial infarction, angina,stroke, diabetes mellitus (e.g., diabetic nephropathy), renal diseasee.g., chronic kidney disease or diabetic nephropathy optionally in thecontext of pregnancy, renal failure, e.g., chronic renal failure,cognitive dysfunction (such as Alzheimer's), and systemic sclerosis(e.g., scleroderma renal crisis). In certain embodiments, AGT-associateddisease includes intrauterine growth restriction (IUGR) or fetal growthrestriction.

In one embodiment, an iRNA agent targeting AGT is administered to asubject having a AGT-associated disease such that AGT levels, e.g., in acell, tissue, blood or other tissue or fluid of the subject are reducedby at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or morewhen the dsRNA agent is administered to the subject. In preferredembodiments, the AGT level is reduced by at least 20%.

The methods and uses of the invention include administering acomposition described herein such that expression of the target AGT geneis decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18, 24,28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours.In one embodiment, expression of the target AGT gene is decreased for anextended duration, e.g., at least about two, three, four, five, six,seven days or more, e.g., about one week, two weeks, three weeks, orabout four weeks or longer.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with anAGT-associated disease. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. Inpreferred embodiments, the AGT level is reduced by at least 20%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of dyslipidemiahypertension may beassessed, for example, by periodic monitoring of blood pressure.Comparison of the later readings with the initial readings provide aphysician an indication of whether the treatment is effective. It iswell within the ability of one skilled in the art to monitor efficacy oftreatment or prevention by measuring any one of such parameters, or anycombination of parameters. In connection with the administration of aniRNA targeting AGT or pharmaceutical composition thereof, “effectiveagainst” an AGT-associated disease indicates that administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such asimprovement of symptoms, a cure, a reduction in disease, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating aAGT-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed. Suitable animal models ofan angiotensinogen-associated disease, e.g., hypertension, include, forexample, genetic models of hypertension, e.g., BPH/2 mice, sponateouslyhypertensive rats (SHRs), Dahl salt-sensitive rates (DS), TGR(mREN2)27transgenic rats in which endogenous renal renin has been suppressed, andborderline hypertensive rats (BHR), experimentally induced models ofhypertension, e.g., experimentally induced models of renal hypertensionthe Goldblatt model of renal-induced experimental hypertension, subtotalnephrectomy models, and angiotensin II induced hypertension (see, e.g.,Domal and Silva (2011) J Biosci 36:731). Suitable animal models ofpregnancy-associated hypertension include, for example, genetic models,e.g., borderline hypertensive mice (e.g., BPH/5 mice), rats and/or micecarrying a transgene encoding human renin and a transgene encoding humanangiotensinogen, and experimentally induced models, e.g., sFlt-1infusion models, AT1-AA-induced models, reduced uteroplacental perfusionpressure (RUPP) models (see, e.g., McCarthy, et al. (2011) Placenta32:413-419).

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg,1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kgdsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kgdsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kgdsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kgdsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kgdsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kgdsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kgdsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kgdsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kgdsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kgdsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kgdsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kgdsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kgdsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kgdsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kgdsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kgdsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kgdsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kgdsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kgdsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA,30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about50 mg/kg dsRNA. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as weekly,biweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration weekly or biweekly for three months, administrationcan be repeated once per month, for six months or a year or longer.

Administration of the iRNA can reduce AGT levels, e.g., in a cell,tissue, blood, urine or other compartment of the patient by at leastabout 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion, and monitored foradverse effects, such as an allergic reaction. In another example, thepatient can be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on AGT expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, where themodified or unmodified iRNA agent is directly suspended in aqueous orsuitable buffer solvent, as a “free iRNA.” A free iRNA is administeredin the absence of a pharmaceutical composition. The free iRNA may be ina suitable buffer solution. The buffer solution may comprise acetate,citrate, prolamine, carbonate, or phosphate, or any combination thereof.In one embodiment, the buffer solution is phosphate buffered saline(PBS). The pH and osmolarity of the buffer solution containing the iRNAcan be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of AGTgene expression are those having a AGT-associated disease or disorder asdescribed herein.

Treatment of a subject that would benefit from a reduction and/orinhibition of AGT gene expression includes therapeutic and prophylactictreatment.

The invention further provides methods and uses of an iRNA agent or apharmaceutical composition thereof for treating a subject that wouldbenefit from reduction and/or inhibition of AGT expression, e.g., asubject having a AGT-associated disease, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. Forexample, in certain embodiments, an iRNA targeting AGT is administeredin combination with, e.g., an agent useful in treating an AGT-associateddisease as described elsewhere herein. For example, additionaltherapeutics and therapeutic methods suitable for treating a subjectthat would benefit from reduction in AGT expression, e.g., a subjecthaving a AGT-associated disease, include angioplasty, aortorenal bypass,renal denervation, percutaneous transluminal renal angioplasty (PTRA)and stenting, surgical revascularization, catheter-based renalsympathetic denervation, and surgical removal of pheochromocytoma orreninoma, adrenalectomy, treatment with a diuretic, e.g., athiazide-type diuretic, e.g., chlorothiazide, hydrochlorothiazide,chlorthalidone, metolazone, and indapamide, a potassium-sparringdiuretic, such as triamterene and amiloride, a loop diuretic, e.g.,furosemide, torsemide, ethacrynic acid, and bumetanide; an angiotensinconverting enzyme (ACE) inhibitor, e.g., fosinopril, captopril,ramipril, enalapril, lisinopril, and quinapril; an angiotensin IIreceptor antagonist (also known as an angiotensin receptor blocker),e.g., losartan, valsartan, olmesartan, eprosartan, and azilsartan; abeta-blocker, such as a beta-1 selective beta-blocker, e.g., atenolol,metoprolol, propranolol, bisoprolol, and timolol, an alpha-1 receptorbeta-blocker, e.g., labetalol, esmolol, and carvedilol, an intrinsicsympathomimetic beta-blocker, e.g., acebutolol and pindolol; avasodialator, e.g., hydralazine, minoxidil, sodium nitroprusside, andnitroglycerin; a calcium channel blocker, e.g., nifedipine, clevidipine,amlodipine, felodipine, diltiazem, nicardipine, and verapamil; analdosterone antagonist, such as a selective aldosterone antagonist,e.g., eplerenone and spironolactone; an alphaz-agonist, such as acentral-acting alphaz-agonist, e.g., methyldopa, clonidine, andguanfacine, a renin inhibitor, e.g., aliskiren; an alpha-blocker, e.g.,prazosin, terazosin, and doxazosin; a peripheral acting adrenergicagent, e.g., reserpine; a selective D1 receptor partial agonist, e.g.,fenoldopam mesylate; a nonselective alpha-adrenergic antagonist, e.g.,phentolamine; a synthetic, steroidal antimineralocorticoid agent, e.g.,spironolactone, or a combination of any of the foregoing; and atherapeutic agent formulated as a combination of agents, e.g., acombination of amlodipine/benazepril (Lotrel), amlodipine/olmesartan(Azor), amlodipine/telmisartan (Twynsta), amlodipine/valsartan(Exforge), amlodipine/valsartan/hydrochlorothiazide (Exforge HCT),amlodipine/aliskiren (Tekamlo), amlodipine/aliskiren/hydrochlorothiazide(Amturnide), olmesartan/amlodipine/hydrochlorothiazide (Tribenzor),trandolapril/verapamil (Tarka), benazepril/hydrochlorothiazide (LotensinHCT), captopril/hydrochlorothiazide (Capozide),enalapril/hydrochlorothiazide (Vaseretic),fosinopril/hydrochlorothiazide, lisinopril/hydrochlorothiazide(Prinzide, Zestoretic), moexipril/hydrochlorothiazide (Uniretic),quinapril/hydrochlorothiazide (Accuretic),candesartan/hydrochlorothiazide (Atacand HCT),eprosartan/hydrochlorothiazide (Teveten HCT),irbesartan/hydrochlorothiazide (Avalide), losartan/hydrochlorothiazide(Hyzaar), olmesartan/hydrochlorothiazide (Benicar HCT),telmisartan/hydrochlorothiazide (Micardis HCT),valsartan/hydrochlorothiazide (Diovan HCT), atenolol/chlorthalidone(Tenoretic), bisoprolol/hydrochlorothiazide (Ziac),metoprolol/hydrochlorothiazide (Lopressor HCT),nadolol/bendroflumethiazide (Corzide), propranolol/hydrochlorothiazide,aliskiren/hydrochlorothiazide (Tekturna HCT), clonidine/chlorthalidone(Clorpres), spironolactone/hydrochlorothiazide (Aldactazide),triamterene/hydrochlorothiazide (Dyazide, Maxzide),methyldopa/hydrochlorothiazide, and amiloride/hydrochlorothiazide, orother therapeutic agents for treating a AGT-associated disease.

The iRNA agent and an additional therapeutic agent and/or treatment maybe administered at the same time and/or in the same combination, e.g.,parenterally, or the additional therapeutic agent can be administered aspart of a separate composition or at separate times and/or by anothermethod known in the art or described herein.

The present invention also provides methods of using an iRNA agent ofthe invention and/or a composition containing an iRNA agent of theinvention to reduce and/or inhibit AGT expression in a cell. In otheraspects, the present invention provides an iRNA of the invention and/ora composition comprising an iRNA of the invention for use in reducingand/or inhibiting AGT expression in a cell. In yet other aspects, use ofan iRNA of the invention and/or a composition comprising an iRNA of theinvention for the manufacture of a medicament for reducing and/orinhibiting AGT expression in a cell are provided.

The methods and uses include contacting the cell with an iRNA, e.g., adsRNA, of the invention and maintaining the cell for a time sufficientto obtain degradation of the mRNA transcript of an AGT gene, therebyinhibiting expression of the AGT gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of AGT may be determinedby determining the mRNA expression level of AGT using methods routine toone of ordinary skill in the art, e.g., northern blotting, qRT-PCR, bydetermining the protein level of AGT using methods routine to one ofordinary skill in the art, such as western blotting, immunologicaltechniques, flow cytometry methods, ELISA, and/or by determining abiological activity of AGT.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an AGT gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

AGT expression may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%. In preferredembodiments, the AGT level is reduced by at least 20%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the AGT gene of the mammal to be treated. When theorganism to be treated is a human, the composition can be administeredby any means known in the art including, but not limited tosubcutaneous, intravenous, oral, intraperitoneal, or parenteral routes,including intracranial (e.g., intraventricular, intraparenchymal andintrathecal), intramuscular, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by subcutaneousor intravenous infusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof AGT, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

A higher dose may be administered initially (i.e., a loading dose),followed by a lower dosage for a sustained period of time.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an AGT gene in a mammal, e.g., a human. Thepresent invention also provides a composition comprising an iRNA, e.g.,a dsRNA, that targets an AGT gene in a cell of a mammal for use ininhibiting expression of the AGT gene in the mammal. In another aspect,the present invention provides use of an iRNA, e.g., a dsRNA, thattargets an AGT gene in a cell of a mammal in the manufacture of amedicament for inhibiting expression of the AGT gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an iRNA, e.g., a dsRNA, that targets an AGTgene in a cell of the mammal and maintaining the mammal for a timesufficient to obtain degradation of the mRNA transcript of the AGT gene,thereby inhibiting expression of the AGT gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sampleof the iRNA-administered subject by any methods known it the art, e.g.qRT-PCR, described herein. Reduction in protein production can beassessed by any methods known it the art and by methods, e.g., ELISA orwestern blotting, described herein. In one embodiment, a puncture liverbiopsy sample serves as the tissue material for monitoring the reductionin AGT gene and/or protein expression. In another embodiment, a bloodsample serves as the tissue material for monitoring the reduction in AGTgene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target invivo following administration of iRNA agent is done by performing5′-RACE or modifications of the protocol as known in the art (Lasham Aet al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al.(2006) Nature 441: 111-4).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and the Sequence Listing, arehereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Transcripts

siRNA design was carried out to identify siRNAs targeting human (Homosapiens), cynomolgus monkey (Macaca fascicularis; henceforth “cyno”),mouse (Mus musculus), and rat (Rattus norvegicus) AGT transcriptsannotated in the NCBI Gene database (www.ncbi.nlm.nih.gov/gene/). Designused the following transcripts from NCBI: Human —NM_000029.3;Monkey—AB170313.1; Mouse—NM_007428.3. The cynomolgus monkey transcriptwas extended using sequence obtained from a liver-derived cDNA library.Due to high primate/rodent sequence divergence, siRNA duplexes weredesigned in separate batches, including but not limited to batchescontaining duplexes matching human and monkey transcripts only and mousetranscript only. All siRNA duplexes were designed that shared 100%identity to the listed human transcript and other species transcriptsconsidered in each design batch.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were then selected that lacked repeats longerthan seven nucleotides. These 706 candidate human/monkey and 1815 mousesiRNAs were used in comprehensive searches against the appropriatetranscriptomes (defined as the set of NM_ and XM_records within thehuman, monkey, or mouse NCBI Refseq sets) using an exhaustive“brute-force” algorithm implemented in the python script‘BruteForce.py’. The script next parsed the transcript-oligo alignmentsto generate a score based on the position and number of mismatchesbetween the siRNA and any potential “off-target” transcript. Theoff-target score was weighted to emphasize differences in the “seedregion” of siRNAs, in positions 2-9 from the 5′ end of the molecule.Each oligo-transcript pair from the brute-force search was given amismatch score by summing the individual mismatch scores; mismatches inthe position 2-9 were counted as 2.8, mismatches in the cleavage sitepositions 10-11 were counted as 1.2, and mismatches in region 12-19counted as 1.0. An additional off-target prediction was carried out bycomparing the frequency of heptamers and octamers derived from 3distinct, seed-derived hexamers of each oligo. The hexamers frompositions 2-7 relative to the 5′ start is used to create 2 heptamers andone octamer. “Heptamer1” was created by adding a 3′ A to the hexamer;“heptamer2” was created by adding a 5′ A to the hexamer; the octamer wascreated by adding an A to both 5′ and 3′ ends of the hexamer. Thefrequency of octomers and heptamers in the human, monkey, or mouse3′UTRome (defined as the subsequence of the transcriptome from NCBI'sRefseq database where the end of the coding region, the ‘CDS’, isclearly defined) was pre-calculated. The octamer frequency wasnormalized to the heptamer frequency using the median value from therange of octamer frequencies. A “mirSeedScore” was then calculated bycalculating the sum of ((3× normalized octomer count)+(2× heptamer2count)+(1× heptamer1 count)).

Both siRNA strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.The duplexes were sorted by the specificity of the antisense strand.Duplexes from the human/monkey and mouse sets whose antisense oligoslacked GC at the first position, lacked G at both positions 13 and 14,and had 3 or more Us or As in the seed region (characteristics ofduplexes with high predicted efficacy) were selected.

Candidate GalNAc-conjugated duplexes, 21 and 23 nucleotides long on thesense and antisense strands, respectively, were designed by extendingantisense 19mers four additional nucleotides in the 3′ direction(preserving perfect complementarity with the target transcript). Thesense strand was specified as the reverse complement of the first 21nucleotides of the antisense 23mer. Duplexes were selected thatmaintained perfect matches to all selected species transcripts acrossall 23 nucleotides.

siRNA Sequence Selection

A total of 117 sense and 117 antisense derived human/monkey and 42 senseand 42 antisense derived mouse siRNA 19mer oligos were synthesized andformed into GalNAc-conjugated duplexes. A total of 38 sense and 38antisense derived human/monkey 21/23mer oligos and a total of 26 mouse21/23mer oligos were synthesized and formed into GalNAc-conjugatedduplexes.

A detailed list of the unmodified 19mer AGT sense and antisense strandsequences is shown in Table 3.

A detailed list of the modified 19mer AGT sense and antisense strandsequences is shown in Table 4.

A detailed list of the unmodified 21/23mer AGT sense and antisensestrand sequences is shown in Table 7.

A detailed list of the modified 21/23mer AGT sense and antisense strandsequences is shown in Table 8.

A detailed list of unmodified 19mer AGT sense and antisense strandsequences is shown in Table 11.

A detailed list of the unmodified 21/23mer AGT sense and antisensestrand sequences is shown in Table 13.

A detailed list of the modified 21/23mer AGT sense and antisense strandsequences is shown in Table 15.

siRNA Synthesis

General Small and Medium Scale RNA Synthesis Procedure

RNA oligonucleotides are synthesized at scales between 0.2-500 μmolusing commercially available5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and2-N-isobutyrylguanosine and the corresponding 2′-O-methyl and 2′-fluorophosphoramidites according to standard solid phase oligonucleotidesynthesis protocols. The amidite solutions are prepared at 0.1-0.15 Mconcentration and 5-ethylthio-1H-tetrazole (0.25-0.6 M in acetonitrile)is used as the activator. Phosphorothioate backbone modifications areintroduced during synthesis using 0.2 M phenylacetyl disulfide (PADS) inlutidine:acetonitrile (1:1) (v;v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidationstep. After completion of synthesis, the sequences are cleaved from thesolid support and deprotected using methylamine followed bytriethylamine.3HF to remove any 2′-O-t-butyldimethylsilyl protectinggroups present.

For synthesis scales between 5-500 μmol and fully 2′ modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides are deprotected using 3:1 (v/v) ethanol andconcentrated (28-32%) aqueous ammonia either at 35° C. 16 h or 55° C.for 5.5 h. Prior to ammonia deprotection the oligonucleotides aretreated with 0.5 M piperidine in acetonitrile for 20 min on the solidsupport. The crude oligonucleotides are analyzed by LC-MS andanion-exchange HPLC (IEX-HPLC). Purification of the oligonucleotides iscarried out by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH=8.5(buffer A) and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH=8.5 (bufferB). Fractions are analyzed for purity by analytical HPLC. Theproduct-containing fractions with suitable purity are pooled andconcentrated on a rotary evaporator prior to desalting. The samples aredesalted by size exclusion chromatography and lyophilized to dryness.Equal molar amounts of sense and antisense strands are annealed in 1×PBS buffer to prepare the corresponding siRNA duplexes.

For small scales (0.2-1 μmol), synthesis is performed on a MerMade 192synthesizer in a 96 well format. In case of fully 2′-modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides are deprotected using methylamine at room temperaturefor 30-60 min followed by incubation at 60° C. for 30 min or using 3:1(v/v) ethanol and concentrated (28-32%) aqueous ammonia at roomtemperature for 30-60 min followed by incubation at 40° C. for 1.5hours. The crude oligonucleotides are then precipitated in a solution ofacetonitrile:acetone (9:1) and isolated by centrifugation and decantingthe supernatant. The crude oligonucleotide pellet is re-suspended in 20mM NaOAc buffer and analyzed by LC-MS and anion exchange HPLC. The crudeoligonucleotide sequences are desalted in 96 deep well plates on a 5 mLHiTrap Sephadex G25 column (GE Healthcare). In each well about 1.5 mLsamples corresponding to an individual sequence is collected. Thesepurified desalted oligonucleotides are analyzed by LC-MS and anionexchange chromatography. Duplexes are prepared by annealing equimolaramounts of sense and antisense sequences on a Tecan robot. Concentrationof duplexes is adjusted to 10 μM in 1×PBS buffer.

Synthesis of GalNAc-Conjugated Oligonucleotides for In Vivo AnalysisOligonucleotides conjugated with GalNAc ligand at their 3′-terminus aresynthesized at scales between 0.2-500 μmol using a solid supportpre-loaded with a Y-shaped linker bearing a 4,4′-dimethoxytrityl(DMT)-protected primary hydroxy group for oligonucleotide synthesis anda GalNAc ligand attached through a tether.

For synthesis of GalNAc conjugates in the scales between 5-500 μmol, theabove synthesis protocol for RNA is followed with the followingadaptions: For polystyrene-based synthesis supports 5% dichloroaceticacid in toluene is used for DMT-cleavage during synthesis. Cleavage fromthe support and deprotection is performed as described above.Phosphorothioate-rich sequences (usually >5 phorphorothioates) aresynthesized without removing the final 5′-DMT group (“DMT-on”) and,after cleavage and deprotection as described above, purified by reversephase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mMammoniumacetate in 80% acetonitirile (buffer B). Fractions are analyzedfor purity by analytical HPLC and/or LC-MS. The product-containingfractions with suitable purity are pooled and concentrated on a rotaryevaporator. The DMT-group is removed using 20%-25% acetic acid in wateruntil completion. The samples are desalted by size exclusionchromatography and lyophilized to dryness. Equal molar amounts of senseand antisense strands are annealed in 1×PBS buffer to prepare thecorresponding siRNA duplexes.

For small scale synthesis of GalNAc conjugates (0.2-1 μmop, includingsequences with multiple phosphorothioate linkages, the protocolsdescribed above for synthesis of RNA or fully 2′-F/2′-OMe-containingsequences on MerMade platform are applied. Synthesis is performed onpre-packed columns containing GalNAc-functionalized controlled poreglass support.

Example 2. In Vitro Screening of siRNA Duplexes Cell Culture and 96-WellTransfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Cells were washed andre-suspended at 0.125×10⁶ cells/ml. During transfections, cells wereplated onto a 96-well plate with about 20,000 cells per well.

Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μlof Lipofectamine RNAiMax per well (Invitrogen™, Carlsbad Calif. catalognumber 13778-150) to 5 μl of each siRNA duplex to an individual well ina 96-well plate. The mixture was then incubated at room temperature for20 minutes. Eighty μl of complete growth media without antibioticcontaining the appropriate cell number was then added to the siRNAmixture. Cells were incubated for 24 hours prior to RNA purification.

Single dose experiments were performed at 10 nM and 0.01 nM final duplexconcentration. Dose response experiments were done at 10, 1.67, 0.28,0.046, 0.0077, 0.0013, 0.00021, 0.000036 nM final duplex concentration.

Cell Culture and 384-Well Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium(ATCC®) supplemented with 10% FBS, streptomycin, and glutamine (ATCC®)before being released from the plate by trypsinization. Cells werewashed and re-suspended at 0.125×10⁶ cells/ml. During transfections,cells were plated onto a 384-well plate with about 5,000 cells per well.

Transfection was carried out by adding 4.9 μl of Opti-MEM plus 0.1 μl ofLipofectamine RNAiMax per well (Invitrogen™, Carlsbad Calif. catalognumber 13778-150) to 5 μl of each siRNA duplex to an individual well ina 96-well plate. The mixture was then incubated at room temperature for20 minutes. Forty μl of complete growth media without antibioticcontaining the appropriate cell number was then added to the siRNAmixture. Cells were incubated for 24 hours prior to RNA purification.

Single dose experiments were performed at 10 nM and 0.01 nM final duplexconcentration. Dose response experiments were done at 10, 1.67, 0.28,0.046, 0.0077, 0.0013, 0.00021, 0.000036 nM final duplex concentration

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen™,Part #: 610-12) 96-Well Isolation

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for five minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing magnetic stand and the supernatant was removed without disturbingthe beads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were captured again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. Forty μl of supernatantwas removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen™,Part #: 610-12)-384-Well Extraction

Cells were lysed in 50 μl of Lysis/Binding Buffer. Magenetic Dynabeadswere washed in Lysis/Binding Buffer and resuspended in the same. 25 μlLysis/Binding Buffer containing 2 μl of Dynabeads were then added perwell. After shaking plates for 10 minutes at “7” on a Vibratranslator(UnionScientific), an automated plate washing system was utilized(Biotek EL406 with Biostacker, and magnetic capture plate). Plates werethen washed in a manner similar to that described for the 96-wellprocess: twice with buffer A (90 μl), once with buffer B (90 μl), andtwice with buffer C (100 μl). The last wash was removed from the plate,and cDNA synthesis begun immediately.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)—384-WellSynthesis

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 13.2 μl of H₂O perreaction were added to the wells of a 384-well plate containing onlyextracted RNA and magnetic beads (20 μl total volume). cDNA wasgenerated by incubation at 25° C. for 10 min, 37° C. for 120 min, and85° C. for 8 minutes.

Real Time PCR:

Two μl of cDNA were added to a master mix containing 0.5 μl human GAPDHTaqMan Probe (Applied Biosystems Cat #4326317E) and 0.5 μl human AGTTaqMan probe (Applied Biosystems cat # Hs00174854 ml), 24 ofnuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well plates (Roche cat #04887301001).qPCR was performed in a LightCycler 480 real-time PCR machine (Roche).To calculate relative fold change, data were analyzed using the MCtmethod and normalized to assays performed with cells transfected with 10nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4parameter fit model using XLFit and normalized to cells transfected withAD-1955 or mock-transfected.

The sense and antisense sequences of AD-1955 are:

SENSE:  (SEQ ID NO: 13) cuuAcGcuGAGuAcuucGAdTsdT ANTISENSE: (SEQ ID NO: 14) UCGAAGuACUcAGCGuAAGdTsdT.

Table 5 shows the results of a single dose screen in Hep3B cellstransfected using the 96-well method with the indicated 19mer AGT iRNAs.Data are expressed as percent of mRNA remaining relative to untreatedcells.

Table 6 shows the dose response of Hep3B cells transfected using the96-well method with the indicated 19mer AGT iRNAs. The indicated IC₅₀values represent the IC₅₀ values relative to untreated cells.

Table 9 shows the results of a single dose screen in Hep3B cellstransfected using the 384-well method with the indicated 21/23merconjugate AGT iRNAs. Data are expressed as percent of mRNA remainingrelative to untreated cells.

Table 10 shows the dose response of Hep3B cells transfected using the384-well method with the indicated 21/23mer conjugate AGT iRNAs. Theindicated IC₅₀ values represent the IC₅₀ values relative to untreatedcells.

Table 12 shows the results of a single dose screen in Hep3B cellstransfected using the 96-well method with the indicated 19mer AGT iRNAs.Data are expressed as percent of mRNA remaining relative to untreatedcells.

Table 14 shows the results of a single dose, dose-reponse screen of hAGTknockdown with the indicated 21/23mer conjugate AGT iRNAs in miceinfected with an AAV vector expressing hAGT.

Table 16 shows the results of a single dose, dose-reponse screen of hAGTknockdown with the indicated 21/23mer conjugate AGT iRNAs in miceinfected with an AAV vector expressing hAGT.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 (dt) or dT deoxy-thymine dC 2′-deoxycytidine-3′-phosphate Y44inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Tgn)Thymidine-glycol nucleic acid (GNA) S-Isomer P Phosphate VPVinyl-phosphate (Aam) 2′-O-(N-methylacetamide)adenosine-3′-phosphate(Aams) 2′-O-(N-methylacetamide)adenosine-3′-phosphorothioate (Tgn)Thymidine-glycol nucleic acid (GNA) S-Isomer (Cgn) Cytidine-glycolnucleic acid (GNA)

TABLE 3Unmodified Sense and Antisense Strand Sequences of AGT dsRNAs (19mers)Position SEQ Position SEQ relative to ID Antisense relative to IDDuplex Name NM_000029.3 Sense Name Sense Sequence NO Name NM_000029.3Antisense Sequence NO UM AD-56041.1 485-503 A-115161.1UUCUGGGUACUACAGCAGA 15 A-115162.1 485-503 UCUGCUGUAGUACCCAGAA 131UM AD-52431.1 491-509 A-107992.1 GUACUACAGCAGAAGGGUA 16 A-107993.1491-509 UACCCUUCUGCUGUAGUAC 132 UM AD-56057.1 606-624 A-115229.1GUGACCGGGUGUACAUACA 17 A-115230.1 606-624 UGUAUGUACACCCGGUCAC 133UM AD-56047.1 635-653 A-115163.1 CUCGUCAUCCACAAUGAGA 18 A-115164.1635-653 UCUCAUUGUGGAUGACGAG 134 UM AD-52437.1 643-661 A-107994.1CCACAAUGAGAGUACCUGU 19 A-107995.1 643-661 ACAGGUACUCUCAUUGUGG 135UM AD-56064.1 644-662 A-115247.1 CACAAUGAGAGUACCUGUG 20 A-115248.1644-662 CACAGGUACUCUCAUUGUG 136 UM AD-56021.1 658-676 A-115123.1CUGUGAGCAGCUGGCAAAG 21 A-115124.1 658-676 CUUUGCCAGCUGCUCACAG 137UM AD-56067.1 741-759 A-115201.1 CUGUGGAUGAAAAGGCCCU 22 A-115202.1741-759 AGGGCCUUUUCAUCCACAG 138 UM AD-56030.1 822-840 A-115173.1UGGUCGGGAUGCUGGCCAA 23 A-115174.1 822-840 UUGGCCAGCAUCCCGACCA 139UM AD-56034.1 825-843 A-115237.1 UCGGGAUGCUGGCCAACUU 24 A-115238.1825-843 AAGUUGGCCAGCAUCCCGA 140 UM AD-52443.1 828-846 A-107996.1GGAUGCUGGCCAACUUCUU 25 A-107997.1 828-846 AAGAAGUUGGCCAGCAUCC 141UM AD-56017.1 834-852 A-115153.1 UGGCCAACUUCUUGGGCUU 26 A-115154.1834-852 AAGCCCAAGAAGUUGGCCA 142 UM AD-52449.1 841-859 A-107998.1CUUCUUGGGCUUCCGUAUA 27 A-107999.1 841-859 UAUACGGAAGCCCAAGAAG 143UM AD-52455.1 844-862 A-108000.1 CUUGGGCUUCCGUAUAUAU 28 A-108001.1844-862 AUAUAUACGGAAGCCCAAG 144 UM AD-52461.1 849-867 A-108002.1GCUUCCGUAUAUAUGGCAU 29 A-108003.1 849-867 AUGCCAUAUAUACGGAAGC 145UM AD-56025.1 855-873 A-115187.1 GUAUAUAUGGCAUGCACAG 30 A-115188.1855-873 CUGUGCAUGCCAUAUAUAC 146 UM AD-52467.1 863-881 A-108004.1GGCAUGCACAGUGAGCUAU 31 A-108005.1 863-881 AUAGCUCACUGUGCAUGCC 147UM AD-56061.1 878-896 A-115199.1 CUAUGGGGCGUGGUCCAUG 32 A-115200.1878-896 CAUGGACCACGCCCCAUAG 148 UM AD-52473.1 910-928 A-108006.1CUCCCCAACGGCUGUCUUU 33 A-108007.1 910-928 AAAGACAGCCGUUGGGGAG 149UM AD-56063.1 911-929 A-115231.1 UCCCCAACGGCUGUCUUUG 34 A-115232.1911-929 CAAAGACAGCCGUUGGGGA 150 UM AD-56046.1 1002-1020 A-115241.1CUUGGAAGGACAAGAACUG 35 A-115242.1 1002-1020 CAGUUCUUGUCCUUCCAAG 151UM AD-52432.1 1214-1232 A-108008.1 CCACGCUCUCUGGACUUCA 36 A-108009.11214-1232 UGAAGUCCAGAGAGCGUGG 152 UM AD-52438.1 1247-1265 A-108010.1GCUGCUGAGAAGAUUGACA 37 A-108011.1 1247-1265 UGUCAAUCUUCUCAGCAGC 153UM AD-56059.1 1248-1266 A-115167.1 CUGCUGAGAAGAUUGACAG 38 A-115168.11248-1266 CUGUCAAUCUUCUCAGCAG 154 UM AD-56016.1 1249-1267 A-115137.1UGCUGAGAAGAUUGACAGG 39 A-115138.1 1249-1267 CCUGUCAAUCUUCUCAGCA 155UM AD-56068.1 1250-1268 A-115217.1 GCUGAGAAGAUUGACAGGU 40 A-115218.11250-1268 ACCUGUCAAUCUUCUCAGC 156 UM AD-55989.1 1251-1269 A-115081.1CUGAGAAGAUUGACAGGUU 41 A-115082.1 1251-1269 AACCUGUCAAUCUUCUCAG 157UM AD-56040.1 1260-1278 A-115239.1 UUGACAGGUUCAUGCAGGC 42 A-115240.11260-1278 GCCUGCAUGAACCUGUCAA 158 UM AD-56069.1 1277-1295 A-115233.1GCUGUGACAGGAUGGAAGA 43 A-115234.1 1277-1295 UCUUCCAUCCUGUCACAGC 159UM AD-52444.1 1403-1421 A-108012.1 GAGUUCUGGGUGGACAACA 44 A-108013.11403-1421 UGUUGUCCACCCAGAACUC 160 UM AD-56033.1 1408-1426 A-115221.1CUGGGUGGACAACAGCACC 45 A-115222.1 1408-1426 GGUGCUGUUGUCCACCCAG 161UM AD-56058.1 1413-1431 A-115245.1 UGGACAACAGCACCUCAGU 46 A-115246.11413-1431 ACUGAGGUGCUGUUGUCCA 162 UM AD-52450.1 1417-1435 A-108014.1CAACAGCACCUCAGUGUCU 47 A-108015.1 1417-1435 AGACACUGAGGUGCUGUUG 163UM AD-55981.1 1566-1584 A-115141.1 UGGACAAGGUGGAGGGUCU 48 A-115142.11566-1584 AGACCCUCCACCUUGUCCA 164 UM AD-56032.1 1570-1588 A-115205.1CAAGGUGGAGGGUCUCACU 49 A-115206.1 1570-1588 AGUGAGACCCUCCACCUUG 165UM AD-56066.1 1572-1590 A-115185.1 AGGUGGAGGGUCUCACUUU 50 A-115186.11572-1590 AAAGUGAGACCCUCCACCU 166 UM AD-52456.1 1587-1605 A-108016.1CUUUCCAGCAAAACUCCCU 51 A-108017.1 1587-1605 AGGGAGUUUUGCUGGAAAG 167UM AD-56054.1 1591-1609 A-115181.1 CCAGCAAAACUCCCUCAAC 52 A-115182.11591-1609 GUUGAGGGAGUUUUGCUGG 168 UM AD-56035.1 1592-1610 A-115159.1CAGCAAAACUCCCUCAACU 53 A-115160.1 1592-1610 AGUUGAGGGAGUUUUGCUG 169UM AD-52462.1 1595-1613 A-108018.1 CAAAACUCCCUCAACUGGA 54 A-108019.11595-1613 UCCAGUUGAGGGAGUUUUG 170 UM AD-56026.1 1601-1619 A-115203.1UCCCUCAACUGGAUGAAGA 55 A-115204.1 1601-1619 UCUUCAUCCAGUUGAGGGA 171UM AD-56022.1 1602-1620 A-115139.1 CCCUCAACUGGAUGAAGAA 56 A-115140.11602-1620 UUCUUCAUCCAGUUGAGGG 172 UM AD-55983.1 1605-1623 A-115079.1UCAACUGGAUGAAGAAACU 57 A-115080.1 1605-1623 AGUUUCUUCAUCCAGUUGA 173UM AD-56028.1 1728-1746 A-115235.1 CCGAGCUGAACCUGCAAAA 58 A-115236.11728-1746 UUUUGCAGGUUCAGCUCGG 174 UM AD-55980.1 1729-1747 A-115125.1CGAGCUGAACCUGCAAAAA 59 A-115126.1 1729-1747 UUUUUGCAGGUUCAGCUCG 175UM AD-56036.1 1735-1753 A-115175.1 GAACCUGCAAAAAUUGAGC 60 A-115176.11735-1753 GCUCAAUUUUUGCAGGUUC 176 UM AD-56014.1 1737-1755 A-115105.1ACCUGCAAAAAUUGAGCAA 61 A-115106.1 1737-1755 UUGCUCAAUUUUUGCAGGU 177UM AD-56007.1 1738-1756 A-115087.1 CCUGCAAAAAUUGAGCAAU 62 A-115088.11738-1756 AUUGCUCAAUUUUUGCAGG 178 UM AD-56012.1 1739-1757 A-115073.1CUGCAAAAAUUGAGCAAUG 63 A-115074.1 1739-1757 CAUUGCUCAAUUUUUGCAG 179UM AD-56055.1 1740-1758 A-115197.1 UGCAAAAAUUGAGCAAUGA 64 A-115198.11740-1758 UCAUUGCUCAAUUUUUGCA 180 UM AD-56029.1 1741-1759 A-115157.1GCAAAAAUUGAGCAAUGAC 65 A-115158.1 1741-1759 GUCAUUGCUCAAUUUUUGC 181UM AD-52469.1 1767-1785 A-108036.1 GGGUGGGGGAGGUGCUGAA 66 A-108037.11767-1785 UUCAGCACCUCCCCCACCC 182 UM AD-56053.1 1810-1828 A-115165.1GGAUGAGAGAGAGCCCACA 67 A-115166.1 1810-1828 UGUGGGCUCUCUCUCAUCC 183UM AD-52468.1 1879-1897 A-108020.1 CCGCCCAUUCCUGUUUGCU 68 A-108021.11879-1897 AGCAAACAGGAAUGGGCGG 184 UM AD-56045.1 1885-1903 A-115225.1AUUCCUGUUUGCUGUGUAU 69 A-115226.1 1885-1903 AUACACAGCAAACAGGAAU 185UM AD-56056.1 1887-1905 A-115213.1 UCCUGUUUGCUGUGUAUGA 70 A-115214.11887-1905 UCAUACACAGCAAACAGGA 186 UM AD-56010.1 1891-1909 A-115135.1GUUUGCUGUGUAUGAUCAA 71 A-115136.1 1891-1909 UUGAUCAUACACAGCAAAC 187UM AD-56015.1 1892-1910 A-115121.1 UUUGCUGUGUAUGAUCAAA 72 A-115122.11892-1910 UUUGAUCAUACACAGCAAA 188 UM AD-56039.1 2070-2088 A-115223.1CCCCCAGUCUCCCACCUUU 73 A-115224.1 2070-2088 AAAGGUGGGAGACUGGGGG 189UM AD-55991.1 2080-2098 A-115113.1 CCCACCUUUUCUUCUAAUG 74 A-115114.12080-2098 CAUUAGAAGAAAAGGUGGG 190 UM AD-52474.1 2081-2099 A-108022.1CCACCUUUUCUUCUAAUGA 75 A-108023.1 2081-2099 UCAUUAGAAGAAAAGGUGG 191UM AD-56024.1 2082-2100 A-115171.1 CACCUUUUCUUCUAAUGAG 76 A-115172.12082-2100 CUCAUUAGAAGAAAAGGUG 192 UM AD-52433.1 2125-2143 A-108024.1GUUUCUCCUUGGUCUAAGU 77 A-108025.1 2125-2143 ACUUAGACCAAGGAGAAAC 193UM AD-56037.1 2199-2217 A-115191.1 UUGCUGGGUUUAUUUUAGA 78 A-115192.12199-2217 UCUAAAAUAAACCCAGCAA 194 UM AD-56049.1 2200-2218 A-115195.1UGCUGGGUUUAUUUUAGAG 79 A-115196.1 2200-2218 CUCUAAAAUAAACCCAGCA 195UM AD-52439.1 2201-2219 A-108026.1 GCUGGGUUUAUUUUAGAGA go A-108027.12201-2219 UCUCUAAAAUAAACCCAGC 196 UM AD-55978.1 2202-2220 A-115093.1CUGGGUUUAUUUUAGAGAA 81 A-115094.1 2202-2220 UUCUCUAAAAUAAACCCAG 197UM AD-55999.1 2203-2221 A-115147.1 UGGGUUUAUUUUAGAGAAU 82 A-115148.12203-2221 AUUCUCUAAAAUAAACCCA 198 UM AD-56050.1 2206-2224 A-115211.1GUUUAUUUUAGAGAAUGGG 83 A-115212.1 2206-2224 CCCAUUCUCUAAAAUAAAC 199UM AD-56031.1 2209-2227 A-115189.1 UAUUUUAGAGAAUGGGGGU 84 A-115190.12209-2227 ACCCCCAUUCUCUAAAAUA 200 UM AD-56027.1 2227-2245 A-115219.1UGGGGAGGCAAGAACCAGU 85 A-115220.1 2227-2245 ACUGGUUCUUGCCUCCCCA 201UM AD-55987.1 2230-2248 A-115143.1 GGAGGCAAGAACCAGUGUU 86 A-115144.12230-2248 AACACUGGUUCUUGCCUCC 202 UM AD-56043.1 2266-2284 A-115193.1UCCAAAAAGAAUUCCAACC 87 A-115194.1 2266-2284 GGUUGGAAUUCUUUUUGGA 203UM AD-56001.1 2268-2286 A-115085.1 CAAAAAGAAUUCCAACCGA 88 A-115086.12268-2286 UCGGUUGGAAUUCUUUUUG 204 UM AD-52445.1 2279-2297 A-108028.1CCAACCGACCAGCUUGUUU 89 A-108029.1 2279-2297 AAACAAGCUGGUCGGUUGG 205UM AD-52451.1 2283-2301 A-108030.1 CCGACCAGCUUGUUUGUGA 90 A-108031.12283-2301 UCACAAACAAGCUGGUCGG 206 UM AD-52457.1 2284-2302 A-108032.1CGACCAGCUUGUUUGUGAA 91 A-108033.1 2284-2302 UUCACAAACAAGCUGGUCG 207UM AD-52463.1 2285-2303 A-108034.1 GACCAGCUUGUUUGUGAAA 92 A-108035.12285-2303 UUUCACAAACAAGCUGGUC 208 UM AD-55982.1 2290-2308 A-115063.1GCUUGUUUGUGAAACAAAA 93 A-115064.1 2290-2308 UUUUGUUUCACAAACAAGC 209UM AD-56019.1 2291-2309 A-115091.1 CUUGUUUGUGAAACAAAAA 94 A-115092.12291-2309 UUUUUGUUUCACAAACAAG 210 UM AD-55988.1 2292-2310 A-115065.1UUGUUUGUGAAACAAAAAA 95 A-115066.1 2292-2310 UUUUUUGUUUCACAAACAA 211UM AD-55994.1 2294-2312 A-115067.1 GUUUGUGAAACAAAAAAGU 96 A-115068.12294-2312 ACUUUUUUGUUUCACAAAC 212 UM AD-56013.1 2296-2314 A-115089.1UUGUGAAACAAAAAAGUGU 97 A-115090.1 2296-2314 ACACUUUUUUGUUUCACAA 213UM AD-56065.1 2299-2317 A-115169.1 UGAAACAAAAAAGUGUUCC 98 A-115170.12299-2317 GGAACACUUUUUUGUUUCA 214 UM AD-56008.1 2304-2322 A-115103.1CAAAAAAGUGUUCCCUUUU 99 A-115104.1 2304-2322 AAAAGGGAACACUUUUUUG 215UM AD-56048.1 2306-2324 A-115179.1 AAAAAGUGUUCCCUUUUCA 100 A-115180.12306-2324 UGAAAAGGGAACACUUUUU 216 UM AD-56003.1 2307-2325 A-115117.1AAAAGUGUUCCCUUUUCAA 101 A-115118.1 2307-2325 UUGAAAAGGGAACACUUUU 217UM AD-56051.1 2314-2332 A-115227.1 UUCCCUUUUCAAGUUGAGA 102 A-115228.12314-2332 UCUCAACUUGAAAAGGGAA 218 UM AD-56044.1 2317-2335 A-115209.1CCUUUUCAAGUUGAGAACA 103 A-115210.1 2317-2335 UGUUCUCAACUUGAAAAGG 219UM AD-55996.1 2320-2338 A-115099.1 UUUCAAGUUGAGAACAAAA 104 A-115100.12320-2338 UUUUGUUCUCAACUUGAAA 220 UM AD-56002.1 2321-2339 A-115101.1UUCAAGUUGAGAACAAAAA 105 A-115102.1 2321-2339 UUUUUGUUCUCAACUUGAA 221UM AD-55976.1 2323-2341 A-115061.1 CAAGUUGAGAACAAAAAUU 106 A-115062.12323-2341 AAUUUUUGUUCUCAACUUG 222 UM AD-56062.1 2325-2343 A-115215.1AGUUGAGAACAAAAAUUGG 107 A-115216.1 2325-2343 CCAAUUUUUGUUCUCAACU 223UM AD-56011.1 2326-2344 A-115151.1 GUUGAGAACAAAAAUUGGG 108 A-115152.12326-2344 CCCAAUUUUUGUUCUCAAC 224 UM AD-56009.1 2328-2346 A-115119.1UGAGAACAAAAAUUGGGUU 109 A-115120.1 2328-2346 AACCCAAUUUUUGUUCUCA 225UM AD-56020.1 2329-2347 A-115107.1 GAGAACAAAAAUUGGGUUU 110 A-115108.12329-2347 AAACCCAAUUUUUGUUCUC 226 UM AD-55979.1 2331-2349 A-115109.1GAACAAAAAUUGGGUUUUA 111 A-115110.1 2331-2349 UAAAACCCAAUUUUUGUUC 227UM AD-55997.1 2332-2350 A-115115.1 AACAAAAAUUGGGUUUUAA 112 A-115116.12332-2350 UUAAAACCCAAUUUUUGUU 228 UM AD-56000.1 2333-2351 A-115069.1ACAAAAAUUGGGUUUUAAA 113 A-115070.1 2333-2351 UUUAAAACCCAAUUUUUGU 229UM AD-56006.1 2334-2352 A-115071.1 CAAAAAUUGGGUUUUAAAA 114 A-115072.12334-2352 UUUUAAAACCCAAUUUUUG 230 UM AD-55990.1 2339-2357 A-115097.1AUUGGGUUUUAAAAUUAAA 115 A-115098.1 2339-2357 UUUAAUUUUAAAACCCAAU 231UM AD-55984.1 2340-2358 A-115095.1 UUGGGUUUUAAAAUUAAAG 116 A-115096.12340-2358 CUUUAAUUUUAAAACCCAA 232 UM AD-55995.1 2341-2359 A-115083.1UGGGUUUUAAAAUUAAAGU 117 A-115084.1 2341-2359 ACUUUAAUUUUAAAACCCA 233UM AD-56004.1 2346-2364 A-115133.1 UUUAAAAUUAAAGUAUACA 118 A-115134.12346-2364 UGUAUACUUUAAUUUUAAA 234 UM AD-56042.1 2347-2365 A-115177.1UUAAAAUUAAAGUAUACAU 119 A-115178.1 2347-2365 AUGUAUACUUUAAUUUUAA 235UM AD-55992.1 2384-2402 A-115129.1 UUGUAUUUAGUGUCUUGAA 120 A-115130.12384-2402 UUCAAGACACUAAAUACAA 236 UM AD-55977.1 2385-2403 A-115077.1UGUAUUUAGUGUCUUGAAU 121 A-115078.1 2385-2403 AUUCAAGACACUAAAUACA 237UM AD-56005.1 2391-2409 A-115149.1 UAGUGUCUUGAAUGUAAGA 122 A-115150.12391-2409 UCUUACAUUCAAGACACUA 238 UM AD-56052.1 2397-2415 A-115243.1CUUGAAUGUAAGAACAUGA 123 A-115244.1 2397-2415 UCAUGUUCUUACAUUCAAG 239UM AD-55985.1 2398-2416 A-115111.1 UUGAAUGUAAGAACAUGAC 124 A-115112.12398-2416 GUCAUGUUCUUACAUUCAA 240 UM AD-56038.1 2439-2457 A-115207.1CUUAGUUUUUUCCACAGAU 125 A-115208.1 2439-2457 AUCUGUGGAAAAAACUAAG 241UM AD-56023.1 2450-2468 A-115155.1 CCACAGAUGCUUGUGAUUU 126 A-115156.12450-2468 AAAUCACAAGCAUCUGUGG 242 UM AD-56018.1 2452-2470 A-115075.1ACAGAUGCUUGUGAUUUUU 127 A-115076.1 2452-2470 AAAAAUCACAAGCAUCUGU 243UM AD-56060.1 2497-2515 A-115183.1 CUGAAUUUCUGUUUGAAUG 128 A-115184.12497-2515 CAUUCAAACAGAAAUUCAG 244 UM AD-55993.1 2519-2537 A-115145.1AACCAUAGCUGGUUAUUUC 129 A-115146.1 2519-2537 GAAAUAACCAGCUAUGGUU 245UM AD-55986.1 2521-2539 A-115127.1 CCAUAGCUGGUUAUUUCUC 130 A-115128.12521-2539 GAGAAAUAACCAGCUAUGG 246

TABLE 4Modified Sense and Antisense Strand Sequences of AGT dsRNAs (19 mers)SEQ Anti- SEQ Duplex Sense ID sense  ID Name Name Sense Sequence NO.Name Antisense Sequence NO. AD-56041.1 A-115161.1uucuGGGuAcuAcAGcAGAdTsdT 247 A-115162.1 UCUGCUGuAGuACCcAGAAdTsdT 363AD-52431.1 A-107992.1 GuAcuAcAGcAGAAGGGuAdTsdT 248 A-107993.1uACCCUUCUGCUGuAGuACdTsdT 364 AD-56057.1 A-115229.1GuGAccGGGuGuAcAuAcAdTsdT 249 A-115230.1 UGuAUGuAcACCCGGUcACdTsdT 365AD-56047.1 A-115163.1 cucGucAuccAcAAuGAGAdTsdT 250 A-115164.1UCUcAUUGUGGAUGACGAGdTsdT 366 AD-52437.1 A-107994.1ccAcAAuGAGAGuAccuGudTsdT 251 A-107995.1 AcAGGuACUCUcAUUGUGGdTsdT 367AD-56064.1 A-115247.1 cAcAAuGAGAGuAccuGuGdTsdT 252 A-115248.1cAcAGGuACUCUcAUUGUGdTsdT 368 AD-56021.1 A-115123.1cuGuGAGcAGcuGGcAAAGdTsdT 253 A-115124.1 CUUUGCcAGCUGCUcAcAGdTsdT 369AD-56067.1 A-115201.1 cuGuGGAuGAAAAGGcccudTsdT 254 A-115202.1AGGGCCUUUUcAUCcAcAGdTsdT 370 AD-56030.1 A-115173.1uGGucGGGAuGcuGGccAAdTsdT 255 A-115174.1 UUGGCcAGcAUCCCGACcAdTsdT 371AD-56034.1 A-115237.1 ucGGGAuGcuGGccAAcuudTsdT 256 A-115238.1AAGUUGGCcAGcAUCCCGAdTsdT 372 AD-52443.1 A-107996.1GGAuGcuGGccAAcuucuudTsdT 257 A-107997.1 AAGAAGUUGGCcAGcAUCCdTsdT 373AD-56017.1 A-115153.1 uGGccAAcuucuuGGGcuudTsdT 258 A-115154.1AAGCCcAAGAAGUUGGCcAdTsdT 374 AD-52449.1 A-107998.1cuucuuGGGcuuccGuAuAdTsdT 259 A-107999.1 uAuACGGAAGCCcAAGAAGdTsdT 375AD-52455.1 A-108000.1 cuuGGGcuuccGuAuAuAudTsdT 260 A-108001.1AuAuAuACGGAAGCCcAAGdTsdT 376 AD-52461.1 A-108002.1GcuuccGuAuAuAuGGcAudTsdT 261 A-108003.1 AUGCcAuAuAuACGGAAGCdTsdT 377AD-56025.1 A-115187.1 GuAuAuAuGGcAuGcAcAGdTsdT 262 A-115188.1CUGUGcAUGCcAuAuAuACdTsdT 378 AD-52467.1 A-108004.1GGcAuGcAcAGuGAGcuAudTsdT 263 A-108005.1 AuAGCUcACUGUGcAUGCCdTsdT 379AD-56061.1 A-115199.1 cuAuGGGGcGuGGuccAuGdTsdT 264 A-115200.1cAUGGACcACGCCCcAuAGdTsdT 380 AD-52473.1 A-108006.1cuccccAAcGGcuGucuuudTsdT 265 A-108007.1 AAAGAcAGCCGUUGGGGAGdTsdT 381AD-56063.1 A-115231.1 uccccAAcGGcuGucuuuGdTsdT 266 A-115232.1cAAAGAcAGCCGUUGGGGAdTsdT 382 AD-56046.1 A-115241.1cuuGGAAGGAcAAGAAcuGdTsdT 267 A-115242.1 cAGUUCUUGUCCUUCcAAGdTsdT 383AD-52432.1 A-108008.1 ccAcGcucucuGGAcuucAdTsdT 268 A-108009.1UGAAGUCcAGAGAGCGUGGdTsdT 384 AD-52438.1 A-108010.1GcuGcuGAGAAGAuuGAcAdTsdT 269 A-108011.1 UGUcAAUCUUCUcAGcAGCdTsdT 385AD-56059.1 A-115167.1 cuGcuGAGAAGAuuGAcAGdTsdT 270 A-115168.1CUGUcAAUCUUCUcAGcAGdTsdT 386 AD-56016.1 A-115137.1uGcuGAGAAGAuuGAcAGGdTsdT 271 A-115138.1 CCUGUcAAUCUUCUcAGcAdTsdT 387AD-56068.1 A-115217.1 GcuGAGAAGAuuGAcAGGudTsdT 272 A-115218.1ACCUGUcAAUCUUCUcAGCdTsdT 388 AD-55989.1 A-115081.1cuGAGAAGAuuGAcAGGuudTsdT 273 A-115082.1 AACCUGUcAAUCUUCUcAGdTsdT 389AD-56040.1 A-115239.1 uuGAcAGGuucAuGcAGGcdTsdT 274 A-115240.1GCCUGcAUGAACCUGUcAAdTsdT 390 AD-56069.1 A-115233.1GcuGuGAcAGGAuGGAAGAdTsdT 275 A-115234.1 UCUUCcAUCCUGUcAcAGCdTsdT 391AD-52444.1 A-108012.1 GAGuucuGGGuGGAcAAcAdTsdT 276 A-108013.1UGUUGUCcACCcAGAACUCdTsdT 392 AD-56033.1 A-115221.1cuGGGuGGAcAAcAGcAccdTsdT 277 A-115222.1 GGUGCUGUUGUCcACCcAGdTsdT 393AD-56058.1 A-115245.1 uGGAcAAcAGcAccucAGudTsdT 278 A-115246.1ACUGAGGUGCUGUUGUCcAdTsdT 394 AD-52450.1 A-108014.1cAAcAGcAccucAGuGucudTsdT 279 A-108015.1 AGAcACUGAGGUGCUGUUGdTsdT 395AD-55981.1 A-115141.1 uGGAcAAGGuGGAGGGucudTsdT 280 A-115142.1AGACCCUCcACCUUGUCcAdTsdT 396 AD-56032.1 A-115205.1cAAGGuGGAGGGucucAcudTsdT 281 A-115206.1 AGUGAGACCCUCcACCUUGdTsdT 397AD-56066.1 A-115185.1 AGGuGGAGGGucucAcuuudTsdT 282 A-115186.1AAAGUGAGACCCUCcACCUdTsdT 398 AD-52456.1 A-108016.1cuuuccAGcAAAAcucccudTsdT 283 A-108017.1 AGGGAGUUUUGCUGGAAAGdTsdT 399AD-56054.1 A-115181.1 ccAGcAAAAcucccucAAcdTsdT 284 A-115182.1GUUGAGGGAGUUUUGCUGGdTsdT 400 AD-56035.1 A-115159.1cAGcAAAAcucccucAAcudTsdT 285 A-115160.1 AGUUGAGGGAGUUUUGCUGdTsdT 401AD-52462.1 A-108018.1 cAAAAcucccucAAcuGGAdTsdT 286 A-108019.1UCcAGUUGAGGGAGUUUUGdTsdT 402 AD-56026.1 A-115203.1ucccucAAcuGGAuGAAGAdTsdT 287 A-115204.1 UCUUcAUCcAGUUGAGGGAdTsdT 403AD-56022.1 A-115139.1 cccucAAcuGGAuGAAGAAdTsdT 288 A-115140.1UUCUUcAUCcAGUUGAGGGdTsdT 404 AD-55983.1 A-115079.1ucAAcuGGAuGAAGAAAcudTsdT 289 A-115080.1 AGUUUCUUcAUCcAGUUGAdTsdT 405AD-56028.1 A-115235.1 ccGAGcuGAAccuGcAAAAdTsdT 290 A-115236.1UUUUGcAGGUUcAGCUCGGdTsdT 406 AD-55980.1 A-115125.1cGAGcuGAAccuGcAAAAAdTsdT 291 A-115126.1 UUUUUGcAGGUUcAGCUCGdTsdT 407AD-56036.1 A-115175.1 GAAccuGcAAAAAuuGAGcdTsdT 292 A-115176.1GCUcAAUUUUUGcAGGUUCdTsdT 408 AD-56014.1 A-115105.1AccuGcAAAAAuuGAGcAAdTsdT 293 A-115106.1 UUGCUcAAUUUUUGcAGGUdTsdT 409AD-56007.1 A-115087.1 ccuGcAAAAAuuGAGcAAudTsdT 294 A-115088.1AUUGCUcAAUUUUUGcAGGdTsdT 410 AD-56012.1 A-115073.1cuGcAAAAAuuGAGcAAuGdTsdT 295 A-115074.1 cAUUGCUcAAUUUUUGcAGdTsdT 411AD-56055.1 A-115197.1 uGcAAAAAuuGAGcAAuGAdTsdT 296 A-115198.1UcAUUGCUcAAUUUUUGcAdTsdT 412 AD-56029.1 A-115157.1GcAAAAAuuGAGcAAuGAcdTsdT 297 A-115158.1 GUcAUUGCUcAAUUUUUGCdTsdT 413AD-52469.1 A-108036.1 GGGuGGGGGAGGuGcuGAAdTsdT 298 A-108037.1UUcAGcACCUCCCCcACCCdTsdT 414 AD-56053.1 A-115165.1GGAuGAGAGAGAGcccAcAdTsdT 299 A-115166.1 UGUGGGCUCUCUCUcAUCCdTsdT 415AD-52468.1 A-108020.1 ccGcccAuuccuGuuuGcudTsdT 300 A-108021.1AGcAAAcAGGAAUGGGCGGdTsdT 416 AD-56045.1 A-115225.1AuuccuGuuuGcuGuGuAudTsdT 301 A-115226.1 AuAcAcAGcAAAcAGGAAUdTsdT 417AD-56056.1 A-115213.1 uccuGuuuGcuGuGuAuGAdTsdT 302 A-115214.1UcAuAcAcAGcAAAcAGGAdTsdT 418 AD-56010.1 A-115135.1GuuuGcuGuGuAuGAucAAdTsdT 303 A-115136.1 UUGAUcAuAcAcAGcAAACdTsdT 419AD-56015.1 A-115121.1 uuuGcuGuGuAuGAucAAAdTsdT 304 A-115122.1UUUGAUcAuAcAcAGcAAAdTsdT 420 AD-56039.1 A-115223.1cccccAGucucccAccuuudTsdT 305 A-115224.1 AAAGGUGGGAGACUGGGGGdTsdT 421AD-55991.1 A-115113.1 cccAccuuuucuucuAAuGdTsdT 306 A-115114.1cAUuAGAAGAAAAGGUGGGdTsdT 422 AD-52474.1 A-108022.1ccAccuuuucuucuAAuGAdTsdT 307 A-108023.1 UcAUuAGAAGAAAAGGUGGdTsdT 423AD-56024.1 A-115171.1 cAccuuuucuucuAAuGAGdTsdT 308 A-115172.1CUcAUuAGAAGAAAAGGUGdTsdT 424 AD-52433.1 A-108024.1GuuucuccuuGGucuAAGudTsdT 309 A-108025.1 ACUuAGACcAAGGAGAAACdTsdT 425AD-56037.1 A-115191.1 uuGcuGGGuuuAuuuuAGAdTsdT 310 A-115192.1UCuAAAAuAAACCcAGcAAdTsdT 426 AD-56049.1 A-115195.1uGcuGGGuuuAuuuuAGAGdTsdT 311 A-115196.1 CUCuAAAAuAAACCcAGcAdTsdT 427AD-52439.1 A-108026.1 GcuGGGuuuAuuuuAGAGAdTsdT 312 A-108027.1UCUCuAAAAuAAACCcAGCdTsdT 428 AD-55978.1 A-115093.1cuGGGuuuAuuuuAGAGAAdTsdT 313 A-115094.1 UUCUCuAAAAuAAACCcAGdTsdT 429AD-55999.1 A-115147.1 uGGGuuuAuuuuAGAGAAudTsdT 314 A-115148.1AUUCUCuAAAAuAAACCcAdTsdT 430 AD-56050.1 A-115211.1GuuuAuuuuAGAGAAuGGGdTsdT 315 A-115212.1 CCcAUUCUCuAAAAuAAACdTsdT 431AD-56031.1 A-115189.1 uAuuuuAGAGAAuGGGGGudTsdT 316 A-115190.1ACCCCcAUUCUCuAAAAuAdTsdT 432 AD-56027.1 A-115219.1uGGGGAGGcAAGAAccAGudTsdT 317 A-115220.1 ACUGGUUCUUGCCUCCCcAdTsdT 433AD-55987.1 A-115143.1 GGAGGcAAGAAccAGuGuudTsdT 318 A-115144.1AAcACUGGUUCUUGCCUCCdTsdT 434 AD-56043.1 A-115193.1uccAAAAAGAAuuccAAccdTsdT 319 A-115194.1 GGUUGGAAUUCUUUUUGGAdTsdT 435AD-56001.1 A-115085.1 cAAAAAGAAuuccAAccGAdTsdT 320 A-115086.1UCGGUUGGAAUUCUUUUUGdTsdT 436 AD-52445.1 A-108028.1ccAAccGAccAGcuuGuuudTsdT 321 A-108029.1 AAAcAAGCUGGUCGGUUGGdTsdT 437AD-52451.1 A-108030.1 ccGAccAGcuuGuuuGuGAdTsdT 322 A-108031.1UcAcAAAcAAGCUGGUCGGdTsdT 438 AD-52457.1 A-108032.1cGAccAGcuuGuuuGuGAAdTsdT 323 A-108033.1 UUcAcAAAcAAGCUGGUCGdTsdT 439AD-52463.1 A-108034.1 GAccAGcuuGuuuGuGAAAdTsdT 324 A-108035.1UUUcAcAAAcAAGCUGGUCdTsdT 440 AD-55982.1 A-115063.1GcuuGuuuGuGAAAcAAAAdTsdT 325 A-115064.1 UUUUGUUUcAcAAAcAAGCdTsdT 441AD-56019.1 A-115091.1 cuuGuuuGuGAAAcAAAAAdTsdT 326 A-115092.1UUUUUGUUUcAcAAAcAAGdTsdT 442 AD-55988.1 A-115065.1uuGuuuGuGAAAcAAAAAAdTsdT 327 A-115066.1 UUUUUUGUUUcAcAAAcAAdTsdT 443AD-55994.1 A-115067.1 GuuuGuGAAAcAAAAAAGudTsdT 328 A-115068.1ACUUUUUUGUUUcAcAAACdTsdT 444 AD-56013.1 A-115089.1uuGuGAAAcAAAAAAGuGudTsdT 329 A-115090.1 AcACUUUUUUGUUUcAcAAdTsdT 445AD-56065.1 A-115169.1 uGAAAcAAAAAAGuGuuccdTsdT 330 A-115170.1GGAAcACUUUUUUGUUUcAdTsdT 446 AD-56008.1 A-115103.1cAAAAAAGuGuucccuuuudTsdT 331 A-115104.1 AAAAGGGAAcACUUUUUUGdTsdT 447AD-56048.1 A-115179.1 AAAAAGuGuucccuuuucAdTsdT 332 A-115180.1UGAAAAGGGAAcACUUUUUdTsdT 448 AD-56003.1 A-115117.1AAAAGuGuucccuuuucAAdTsdT 333 A-115118.1 UUGAAAAGGGAAcACUUUUdTsdT 449AD-56051.1 A-115227.1 uucccuuuucAAGuuGAGAdTsdT 334 A-115228.1UCUcAACUUGAAAAGGGAAdTsdT 459 AD-56044.1 A-115209.1ccuuuucAAGuuGAGAAcAdTsdT 335 A-115210.1 UGUUCUcAACUUGAAAAGGdTsdT 451AD-55996.1 A-115099.1 uuucAAGuuGAGAAcAAAAdTsdT 336 A-115100.1UUUUGUUCUcAACUUGAAAdTsdT 452 AD-56002.1 A-115101.1uucAAGuuGAGAAcAAAAAdTsdT 337 A-115102.1 UUUUUGUUCUcAACUUGAAdTsdT 453AD-55976.1 A-115061.1 cAAGuuGAGAAcAAAAAuudTsdT 338 A-115062.1AAUUUUUGUUCUcAACUUGdTsdT 454 AD-56062.1 A-115215.1AGuuGAGAAcAAAAAuuGGdTsdT 339 A-115216.1 CcAAUUUUUGUUCUcAACUdTsdT 455AD-56011.1 A-115151.1 GuuGAGAAcAAAAAuuGGGdTsdT 340 A-115152.1CCcAAUUUUUGUUCUcAACdTsdT 456 AD-56009.1 A-115119.1uGAGAAcAAAAAuuGGGuudTsdT 341 A-115120.1 AACCcAAUUUUUGUUCUcAdTsdT 457AD-56020.1 A-115107.1 GAGAAcAAAAAuuGGGuuudTsdT 342 A-115108.1AAACCcAAUUUUUGUUCUCdTsdT 458 AD-55979.1 A-115109.1GAAcAAAAAuuGGGuuuuAdTsdT 343 A-115110.1 uAAAACCcAAUUUUUGUUCdTsdT 459AD-55997.1 A-115115.1 AAcAAAAAuuGGGuuuuAAdTsdT 344 A-115116.1UuAAAACCcAAUUUUUGUUdTsdT 460 AD-56000.1 A-115069.1AcAAAAAuuGGGuuuuAAAdTsdT 345 A-115070.1 UUuAAAACCcAAUUUUUGUdTsdT 461AD-56006.1 A-115071.1 cAAAAAuuGGGuuuuAAAAdTsdT 346 A-115072.1UUUuAAAACCcAAUUUUUGdTsdT 462 AD-55990.1 A-115097.1AuuGGGuuuuAAAAuuAAAdTsdT 347 A-115098.1 UUuAAUUUuAAAACCcAAUdTsdT 463AD-55984.1 A-115095.1 uuGGGuuuuAAAAuuAAAGdTsdT 348 A-115096.1CUUuAAUUUuAAAACCcAAdTsdT 464 AD-55995.1 A-115083.1uGGGuuuuAAAAuuAAAGudTsdT 349 A-115084.1 ACUUuAAUUUuAAAACCcAdTsdT 465AD-56004.1 A-115133.1 uuuAAAAuuAAAGuAuAcAdTsdT 350 A-115134.1UGuAuACUUuAAUUUuAAAdTsdT 466 AD-56042.1 A-115177.1uuAAAAuuAAAGuAuAcAudTsdT 351 A-115178.1 AUGuAuACUUuAAUUUuAAdTsdT 467AD-55992.1 A-115129.1 uuGuAuuuAGuGucuuGAAdTsdT 352 A-115130.1UUcAAGAcACuAAAuAcAAdTsdT 468 AD-55977.1 A-115077.1uGuAuuuAGuGucuuGAAudTsdT 353 A-115078.1 AUUcAAGAcACuAAAuAcAdTsdT 469AD-56005.1 A-115149.1 uAGuGucuuGAAuGuAAGAdTsdT 354 A-115150.1UCUuAcAUUcAAGAcACuAdTsdT 470 AD-56052.1 A-115243.1cuuGAAuGuAAGAAcAuGAdTsdT 355 A-115244.1 UcAUGUUCUuAcAUUcAAGdTsdT 471AD-55985.1 A-115111.1 uuGAAuGuAAGAAcAuGAcdTsdT 356 A-115112.1GUcAUGUUCUuAcAUUcAAdTsdT 472 AD-56038.1 A-115207.1cuuAGuuuuuuccAcAGAudTsdT 357 A-115208.1 AUCUGUGGAAAAAACuAAGdTsdT 473AD-56023.1 A-115155.1 ccAcAGAuGcuuGuGAuuudTsdT 358 A-115156.1AAAUcAcAAGcAUCUGUGGdTsdT 474 AD-56018.1 A-115075.1AcAGAuGcuuGuGAuuuuudTsdT 359 A-115076.1 AAAAAUcAcAAGcAUCUGUdTsdT 475AD-56060.1 A-115183.1 cuGAAuuucuGuuuGAAuGdTsdT 360 A-115184.1cAUUcAAAcAGAAAUUcAGdTsdT 476 AD-55993.1 A-115145.1AAccAuAGcuGGuuAuuucdTsdT 361 A-115146.1 GAAAuAACcAGCuAUGGUUdTsdT 477AD-55986.1 A-115127.1 ccAuAGcuGGuuAuuucucdTsdT 362 A-115128.1GAGAAAuAACcAGCuAUGGdTsdT 478

TABLE 5 AGT single dose screen in Hep3B cells Start relative 10 nM 0.1nM 0.1 nM 0.01 nM 0.01 nM to Avg 10 nM SD Avg SD Avg SD NM_000029.3AD-52457.1 9.4 0.6 13.3 1.3 15.1 2.3 2284 AD-52438.1 3.4 0.1 12.2 1.817.7 1.7 1247 AD-52463.1 9.8 0.9 16.9 2.9 19.3 2.1 2285 AD-52433.1 7.41.5 14.4 1.0 24.8 1.3 2125 AD-52439.1 15.1 1.1 23.9 0.2 29.9 1.4 2201AD-52449.1 5.7 0.6 22.7 1.2 42.4 5.3 841 AD-52451.1 10.4 0.5 29.0 1.147.4 1.1 2283 AD-52474.1 5.0 0.3 30.6 2.2 49.1 1.9 2081 AD-52462.1 6.10.1 23.7 2.7 50.8 1.6 1595 AD-52445.1 11.0 0.3 24.2 1.4 55.1 0.4 2279AD-52456.1 11.9 0.3 38.7 0.5 71.7 1.4 1587 AD-52469.1 15.9 0.6 40.6 3.184.1 0.8 1767 AD-52461.1 62.8 1.1 78.5 0.3 85.0 0.6 849 AD-52443.1 16.90.0 49.4 2.0 88.4 3.0 828 AD-52468.1 27.6 1.5 85.1 3.7 91.6 1.4 1879AD-52431.1 24.6 3.7 75.2 1.8 92.3 3.1 491 AD-52444.1 67.0 2.8 74.4 0.394.1 0.2 1403 AD-52455.1 57.0 0.5 89.5 4.1 94.4 1.6 844 AD-52432.1 70.62.3 93.6 8.2 95.7 3.7 1214 AD-52473.1 46.4 0.2 75.6 0.2 95.8 0.1 910AD-52450.1 20.3 1.7 53.0 2.5 96.3 4.8 1417 AD-52437.1 36.3 4.1 86.6 0.696.6 0.9 643 AD-52467.1 59.5 0.8 94.5 1.7 102.9 5.1 863 AD-55976.1 3.65.8 2323 AD-55977.1 77.2 70.0 2385 AD-55978.1 8.3 11.8 2202 AD-55979.17.7 9.1 2331 AD-55980.1 14.1 17.4 1729 AD-55981.1 21.2 29.4 1566AD-56023.1 74.1 77.1 2450 AD-56024.1 17.6 27.8 2082 AD-56025.1 66.5 75.0855 AD-56026.1 13.7 20.2 1601 AD-56027.1 16.9 22.2 2227 AD-56028.1 63.874.0 1728 AD-55982.1 22.9 26.1 2290 AD-55983.1 6.2 8.7 1605 AD-55984.152.0 59.9 2340 AD-55985.1 70.1 65.1 2398 AD-55986.1 80.5 79.3 2521AD-55987.1 12.5 15.5 2230 AD-56029.1 51.3 62.2 1741 AD-56030.1 53.3 58.0822 AD-56031.1 38.1 43.7 2204 AD-56032.1 12.6 15.3 1570 AD-56033.1 99.8100.7 1408 AD-56034.1 87.8 93.0 825 AD-55988.1 46.4 49.5 2292 AD-55989.15.8 9.7 1251 AD-55990.1 6.5 5.0 2339 AD-55991.1 50.4 38.2 2080AD-55992.1 76.4 60.5 2384 AD-55993.1 66.5 62.9 2519 AD-56035.1 17.9 17.61592 AD-56036.1 47.7 59.9 1735 AD-56037.1 80.8 83.5 2199 AD-56038.1 84.890.4 2439 AD-56039.1 47.9 66.7 2070 AD-56040.1 95.0 98.3 1260 AD-55994.13.6 5.5 2294 AD-55995.1 31.4 47.8 2341 AD-55996.1 5.1 5.0 2320AD-55997.1 83.3 76.6 2332 AD-55999.1 74.6 75.0 2203 AD-56041.1 21.9 21.4485 AD-56042.1 73.3 73.5 2347 AD-56043.1 53.1 57.8 2266 AD-56044.1 12.013.9 2317 AD-56045.1 41.7 48.8 1885 AD-56046.1 83.0 90.1 1002 AD-56000.113.3 20.3 2333 AD-56001.1 4.7 5.7 2268 AD-56002.1 5.4 6.6 2321AD-56003.1 4.2 4.4 2307 AD-56004.1 54.0 68.5 2346 AD-56005.1 60.2 62.22391 AD-56047.1 24.7 28.2 635 AD-56048.1 7.1 7.4 2306 AD-56049.1 73.062.6 2200 AD-56050.1 96.4 102.1 2206 AD-56051.1 17.4 23.0 2314AD-56052.1 85.1 102.5 2397 AD-56006.1 8.1 10.7 2334 AD-56007.1 17.8 19.11738 AD-56008.1 5.4 4.8 2304 AD-56009.1 12.2 14.4 2328 AD-56010.1 62.169.0 1891 AD-56011.1 35.3 40.3 2326 AD-56053.1 5.0 6.7 1810 AD-56054.130.9 35.5 1591 AD-56055.1 61.4 50.9 1740 AD-56056.1 83.6 94.0 1887AD-56057.1 85.7 97.9 606 AD-56058.1 68.6 87.4 1413 AD-56012.1 35.0 47.11739 AD-56013.1 49.2 57.6 2296 AD-56014.1 54.4 60.3 1737 AD-56015.1 42.246.7 1892 AD-56016.1 11.2 15.6 1249 AD-56017.1 10.6 12.9 834 AD-56059.137.3 45.2 1248 AD-56060.1 94.1 94.3 2497 AD-56061.1 99.8 94.6 878AD-56062.1 19.2 32.2 2325 AD-56063.1 102.2 102.8 911 AD-56064.1 80.292.3 644 AD-56018.1 72.0 74.5 2452 AD-56019.1 25.2 27.7 2291 AD-56020.110.2 15.3 2329 AD-56021.1 36.0 40.9 658 AD-56022.1 37.5 52.7 1602AD-56065.1 56.5 71.9 2299 AD-56066.1 12.3 18.9 1572 AD-56067.1 64.9 81.8741 AD-56068.1 68.6 82.1 1250 AD-56069.1 48.9 72.7 1277

TABLE 6 AGT IC₅₀ data in Hep3B Cells Duplex Name IC₅₀ (nM) AD-524315.719 AD-52432 >10 nM AD-52433 0.057 AD-52437 >10 nM AD-52438 0.063AD-52439 0.192 AD-52443 0.504 AD-52444 >10 nM AD-52445 0.183 AD-524490.211 AD-52450 2.052 AD-52451 0.318 AD-52455 >10 nM AD-52456 1.569AD-52461 >10 nM AD-52462 0.242 AD-52463 0.112 AD-52467 >10 nMAD-52468 >10 nM AD-52469 0.540 AD-52473 >10 nM AD-52474 0.323 AD-559830.017 AD-55983 0.020 AD-55989 0.017 AD-55989 0.019 AD-56016 0.018AD-56016 0.019 AD-56017 0.027 AD-56017 0.031 AD-56053 0.007 AD-560530.009

TABLE 7Unmodified Sense and Antisense Strand Sequences of AGT dsRNAs (21/23 mers)Position relative SEQ Position SEQ to NM_ Sense ID Antisense relative toID Duplex Name 000029.3 Name Sense Sequence NO. Name NM_000029.3Antisense Sequence NO. UM AD-60803.1  830-850 A-122599.1CGGGAUGCUGGCCAACUUCUU 479 A-122600.1  828-850 AAGAAGUUGGCCAGCAUCCCGAC517 UM AD-60775.1  843-863 A-122541.1 AACUUCUUGGGCUUCCGUAUA 480A-122542.1  841-863 UAUACGGAAGCCCAAGAAGUUGG 518 UM AD-60779.1  855-875A-122605.1 UUCCGUAUAUAUGGCAUGCAA 481 A-122606.1  853-875UUGCAUGCCAUAUAUACGGAAGC 519 UM AD-60797.1  857-877 A-122581.1CCGUAUAUAUGGCAUGCACAA 482 A-122582.1  855-877 UUGUGCAUGCCAUAUAUACGGAA520 UM AD-60806.1  880-900 A-122585.1 AGCUAUGGGGCGUGGUCCAUA 483A-122586.1  878-900 UAUGGACCACGCCCCAUAGCUCA 521 UM AD-60787.1  912-932A-122577.1 CUCUCCCCAACGGCUGUCUUU 484 A-122578.1  910-932AAAGACAGCCGUUGGGGAGAGGA 522 UM AD-60807.1  913-933 A-122601.1UCUCCCCAACGGCUGUCUUUA 485 A-122602.1  911-933 UAAAGACAGCCGUUGGGGAGAGG523 UM AD-60794.1 1276-1296 A-122611.1 UGCAGGCUGUGACAGGAUGGA 486A-122612.1 1274-1296 UCCAUCCUGUCACAGCCUGCAUG 524 UM AD-60796.1 1568-1588A-122565.1 CCUGGACAAGGUGGAGGGUCU 487 A-122566.1 1566-1588AGACCCUCCACCUUGUCCAGGUC 525 UM AD-60778.1 1572-1592 A-122589.1GACAAGGUGGAGGGUCUCACU 488 A-122590.1 1570-1592 AGUGAGACCCUCCACCUUGUCCA526 UM AD-60792.1 1574-1594 A-122579.1 CAAGGUGGAGGGUCUCACUUU 489A-122580.1 1572-1594 AAAGUGAGACCCUCCACCUUGUC 527 UM AD-60772.1 1594-1614A-122571.1 UCCAGCAAAACUCCCUCAACU 490 A-122572.1 1592-1614AGUUGAGGGAGUUUUGCUGGAAA 528 UM AD-60773.1 1603-1623 A-122587.1ACUCCCUCAACUGGAUGAAGA 491 A-122588.1 1601-1623 UCUUCAUCCAGUUGAGGGAGUUU529 UM AD-60782.1 1737-1757 A-122575.1 CUGAACCUGCAAAAAUUGAGA 492A-122576.1 1735-1757 UCUCAAUUUUUGCAGGUUCAGCU 530 UM AD-60800.1 1739-1759A-122551.1 GAACCUGCAAAAAUUGAGCAA 493 A-122552.1 1737-1759UUGCUCAAUUUUUGCAGGUUCAG 531 UM AD-60785.1 1741-1761 A-122545.1ACCUGCAAAAAUUGAGCAAUA 494 A-122546.1 1739-1761 UAUUGCUCAAUUUUUGCAGGUUC532 UM AD-60802.1 1742-1762 A-122583.1 CCUGCAAAAAUUGAGCAAUGA 495A-122584.1 1740-1762 UCAUUGCUCAAUUUUUGCAGGUU 533 UM AD-60799.1 1812-1832A-122613.1 GCGGAUGAGAGAGAGCCCACA 496 A-122614.1 1810-1832UGUGGGCUCUCUCUCAUCCGCUU 534 UM AD-60781.1 1894-1914 A-122559.1UGUUUGCUGUGUAUGAUCAAA 497 A-122560.1 1892-1914 UUUGAUCAUACACAGCAAACAGG535 UM AD-60793.1 2072-2092 A-122595.1 CACCCCCAGUCUCCCACCUUU 498A-122596.1 2070-2092 AAAGGUGGGAGACUGGGGGUGAC 536 UM AD-60784.1 2081-2101A-122607.1 UCUCCCACCUUUUCUUCUAAU 499 A-122608.1 2079-2101AUUAGAAGAAAAGGUGGGAGACU 537 UM AD-60777.1 2084-2104 A-122573.1CCCACCUUUUCUUCUAAUGAA 500 A-122574.1 2082-2104 UUCAUUAGAAGAAAAGGUGGGAG538 UM AD-60795.1 2270-2290 A-122549.1 UCCAAAAAGAAUUCCAACCGA 501A-122550.1 2268-2290 UCGGUUGGAAUUCUUUUUGGAAC 539 UM AD-60783.1 2281-2301A-122591.1 UUCCAACCGACCAGCUUGUUU 502 A-122592.1 2279-2301AAACAAGCUGGUCGGUUGGAAUU 540 UM AD-60788.1 2286-2306 A-122593.1ACCGACCAGCUUGUUUGUGAA 503 A-122594.1 2284-2306 UUCACAAACAAGCUGGUCGGUUG541 UM AD-60789.1 2291-2311 A-122609.1 CCAGCUUGUUUGUGAAACAAA 504A-122610.1 2289-2311 UUUGUUUCACAAACAAGCUGGUC 542 UM AD-60770.1 2292-2312A-122539.1 CAGCUUGUUUGUGAAACAAAA 505 A-122540.1 2290-2312UUUUGUUUCACAAACAAGCUGGU 543 UM AD-60776.1 2309-2329 A-122557.1AAAAAAGUGUUCCCUUUUCAA 506 A-122558.1 2307-2329 UUGAAAAGGGAACACUUUUUUGU544 UM AD-60798.1 2316-2336 A-122597.1 UGUUCCCUUUUCAAGUUGAGA 507A-122598.1 2314-2336 UCUCAACUUGAAAAGGGAACACU 545 UM AD-60801.1 2328-2348A-122567.1 AAGUUGAGAACAAAAAUUGGA 508 A-122568.1 2326-2348UCCAAUUUUUGUUCUCAACUUGA 546 UM AD-60791.1 2329-2349 A-122563.1AGUUGAGAACAAAAAUUGGGU 509 A-122564.1 2327-2349 ACCCAAUUUUUGUUCUCAACUUG547 UM AD-60771.1 2334-2354 A-122555.1 AGAACAAAAAUUGGGUUUUAA 510A-122556.1 2332-2354 UUAAAACCCAAUUUUUGUUCUCA 548 UM AD-60780.1 2335-2355A-122543.1 GAACAAAAAUUGGGUUUUAAA 511 A-122544.1 2333-2355UUUAAAACCCAAUUUUUGUUCUC 549 UM AD-60786.1 2386-2406 A-122561.1GUUUGUAUUUAGUGUCUUGAA 512 A-122562.1 2384-2406 UUCAAGACACUAAAUACAAACCG550 UM AD-60790.1 2387-2407 A-122547.1 UUUGUAUUUAGUGUCUUGAAU 513A-122548.1 2385-2407 AUUCAAGACACUAAAUACAAACC 551 UM AD-60774.1 2399-2419A-122603.1 GUCUUGAAUGUAAGAACAUGA 514 A-122604.1 2397-2419UCAUGUUCUUACAUUCAAGACAC 552 UM AD-60804.1 2400-2420 A-122553.1UCUUGAAUGUAAGAACAUGAA 515 A-122554.1 2398-2420 UUCAUGUUCUUACAUUCAAGACA553 UM AD-60805.1 2452-2472 A-122569.1 UUCCACAGAUGCUUGUGAUUU 516A-122570.1 2450-2472 AAAUCACAAGCAUCUGUGGAAAA 554

TABLE 8Modified Sense and Antisense Strand Sequences of AGT dsRNAs (21/23 mers)SEQ Anti- SEQ Duplex Sense ID sense  ID Name Name Sense Sequence NO.Name Antisense Sequence NO. AD-60770.1 A-122539.1CfsasGfcUfuGfuUfUfGfuGfaAfaCfaAfaAfL96 555 A-122540.1usUfsuUfgUfuUfcAfcaaAfcAfaGfcUfgsgsu 593 AD-60771.1 A-122555.1AfsgsAfaCfaAfaAfAfUfuGfgGfuUfuUfaAfL96 556 A-122556.1usUfsaAfaAfcCfcAfauuUfuUfgUfuCfuscsa 594 AD-60772.1 A-122571.1UfscsCfaGfcAfaAfAfCfuCfcCfuCfaAfcUfL96 557 A-122572.1asGfsuUfgAfgGfgAfguuUfuGfcUfgGfasasa 595 AD-60773.1 A-122587.1AfscsUfcCfcUfcAfAfCfuGfgAfuGfaAfgAfL96 558 A-122588.1usCfsuUfcAfuCfcAfguuGfaGfgGfaGfususu 596 AD-60774.1 A-122603.1GfsusCfuUfgAfaUfGfUfaAfgAfaCfaUfgAfL96 559 A-122604.1usCfsaUfgUfuCfuUfacaUfuCfaAfgAfcsasc 597 AD-60775.1 A-122541.1AfsasCfuUfcUfuGfGfGfcUfuCfcGfuAfuAfL96 560 A-122542.1usAfsuAfcGfgAfaGfcccAfaGfaAfgUfusgsg 598 AD-60776.1 A-122557.1AfsasAfaAfaGfuGfUfUfcCfcUfuUfuCfaAfL96 561 A-122558.1usUfsgAfaAfaGfgGfaacAfcUfuUfuUfusgsu 599 AD-60777.1 A-122573.1CfscsCfaCfcUfuUfUfCfuUfcUfaAfuGfaAfL96 562 A-122574.1usUfscAfuUfaGfaAfgaaAfaGfgUfgGfgsasg 600 AD-60778.1 A-122589.1GfsasCfaAfgGfuGfGfAfgGfgUfcUfcAfcUfL96 563 A-122590.1asGfsuGfaGfaCfcCfuccAfcCfuUfgUfcscsa 601 AD-60779.1 A-122605.1UfsusCfcGfuAfuAfUfAfuGfgCfaUfgCfaAfL96 564 A-122606.1usUfsgCfaUfgCfcAfuauAfuAfcGfgAfasgsc 602 AD-60780.1 A-122543.1GfsasAfcAfaAfaAfUfUfgGfgUfuUfuAfaAfL96 565 A-122544.1usUfsuAfaAfaCfcCfaauUfuUfuGfuUfcsusc 603 AD-60781.1 A-122559.1UfsgsUfuUfgCfuGfUfGfuAfuGfaUfcAfaAfL96 566 A-122560.1usUfsuGfaUfcAfuAfcacAfgCfaAfaCfasgsg 604 AD-60782.1 A-122575.1CfsusGfaAfcCfuGfCfAfaAfaAfuUfgAfgAfL96 567 A-122576.1usCfsuCfaAfuUfuUfugcAfgGfuUfcAfgscsu 605 AD-60783.1 A-122591.1UfsusCfcAfaCfcGfAfCfcAfgCfuUfgUfuUfL96 568 A-122592.1asAfsaCfaAfgCfuGfgucGfgUfuGfgAfasusu 606 AD-60784.1 A-122607.1UfscsUfcCfcAfcCfUfUftfacUfuCfuAfaUfL96 569 A-122608.1asUfsuAfgAfaGfaAfaagGfuGfgGfaGfascsu 607 AD-60785.1 A-122545.1AfscsCfuGfcAfaAfAfAfuUfgAfgCfaAfuAfL96 570 A-122546.1usAfsuUfgCfuCfaAfuuuUfuGfcAfgGfususc 608 AD-60786.1 A-122561.1GfsusUfuGfuAfuUfUfAfgUfgUfcUfuGfaAfL96 571 A-122562.1usUfscAfaGfaCfaCfuaaAfuAfcAfaAfcscsg 609 AD-60787.1 A-122577.1CfsusCfuCfcCfcAfAfCfgGfcUfgUfcUfuUfL96 572 A-122578.1asAfsaGfaCfaGfcCfguuGfgGfgAfgAfgsgsa 610 AD-60788.1 A-122593.1AfscsCfgAfcCfaGfCfUfuGfuUfuGfuGfaAfL96 573 A-122594.1usUfscAfcAfaAfcAfagcUfgGfuCfgGfususg 611 AD-60789.1 A-122609.1CfscsAfgCfuUfgUfUfUfgUfgAfaAfcAfaAfL96 574 A-122610.1usUfsuGfuUfuCfaCfaaaCfaAfgCfuGfgsusc 612 AD-60790.1 A-122547.1UfsusUfgUfaUfuUfAfGfuGfuCfuUfgAfaUfL96 575 A-122548.1asUfsuCfaAfgAfcAfcuaAfaUfaCfaAfascsc 613 AD-60791.1 A-122563.1AfsgsUfuGfaGfaAfCfAfaAfaAfuUfgGfgUfL96 576 A-122564.1asCfscCfaAfuUfuUfuguUfcUfcAfaCfususg 614 AD-60792.1 A-122579.1CfsasAfgGfuGfgAfGfGfgUfcUfcAfcUfuUfL96 577 A-122580.1asAfsaGfuGfaGfaCfccuCfcAfcCfuUfgsusc 615 AD-60793.1 A-122595.1CfsasCfcCfcCfaGfUfCfuCfcCfaCfcUfuUfL96 578 A-122596.1asAfsaGfgUfgGfgAfgacUfgGfgGfgUfgsasc 616 AD-60794.1 A-122611.1UfsgsCfaGfgCfuGfUfGfaCfaGfgAfuGfgAfL96 579 A-122612.1usCfscAfuCfcUfgUfcacAfgCfcUfgCfasusg 617 AD-60795.1 A-122549.1UfscsCfaAfaAfaGfAfAfuUfcCfaAfcCfgAfL96 580 A-122550.1usCfsgGfuUfgGfaAfuucUfuUfuUfgGfasasc 618 AD-60796.1 A-122565.1CfscsUfgGfaCfaAfGfGfuGfgAfgGfgUfcUfL96 581 A-122566.1asGfsaCfcCfuCfcAfccuUfgUfcCfaGfgsusc 619 AD-60797.1 A-122581.1CfscsGfuAfuAfuAfUfGfgCfaUfgCfaCfaAfL96 582 A-122582.1usUfsgUfgCfaUfgCfcauAfuAfuAfcGfgsasa 620 AD-60798.1 A-122597.1UfsgsUfuCfcCfuUfUfUfcAfaGfuUfgAfgAfL96 583 A-122598.1usCfsuCfaAfcUfuGfaaaAfgGfgAfaCfascsu 621 AD-60799.1 A-122613.1GfscsGfgAfuGfaGfAfGfaGfaGfcCfcAfcAfL96 584 A-122614.1usGfsuGfgGfcUfcUfcucUfcAfuCfcGfcsusu 622 AD-60800.1 A-122551.1GfsasAfcCfuGfcAfAfAfaAfuUfgAfgCfaAfL96 585 A-122552.1usUfsgCfuCfaAfuUfuuuGfcAfgGfuUfcsasg 623 AD-60801.1 A-122567.1AfsasGfuUfgAfgAfAfCfaAfaAfaUfuGfgAfL96 586 A-122568.1usCfscAfaUfuUfuUfguuCfuCfaAfcUfusgsa 624 AD-60802.1 A-122583.1CfscsUfgCfaAfaAfAfUfuGfaGfcAfaUfgAfL96 587 A-122584.1usCfsaUfuGfcUfcAfauuUfuUfgCfaGfgsusu 625 AD-60803.1 A-122599.1CfsgsGfgAfuGfcUfGfGfcCfaAfcUfuCfuUfL96 588 A-122600.1asAfsgAfaGfuUfgGfccaGfcAfuCfcCfgsasc 626 AD-60804.1 A-122553.1UfscsUfuGfaAfuGfUfAfaGfaAfcAfuGfaAfL96 589 A-122554.1usUfscAfuGfuUfcUfuacAfuUfcAfaGfascsa 627 AD-60805.1 A-122569.1UfsusCfcAfcAfgAfUfGfcUfuGfuGfaUfuUfL96 590 A-122570.1asAfsaUfcAfcAfaGfcauCfuGfuGfgAfasasa 628 AD-60806.1 A-122585.1AfsgsCfuAfuGfgGfGfCfgUfgGfuCfcAfuAfL96 591 A-122586.1usAfsuGfgAfcCfaCfgccCfcAfuAfgCfuscsa 629 AD-60807.1 A-122601.1UfscsUfcCfcCfaAfCfGfgCfuGfuCfuUfuAfL96 592 A-122602.1usAfsaAfgAfcAfgCfcguUfgGfgGfaGfasgsg 630

TABLE 9 AGT single dose screen in Hep3B cells (21/23 mers) Startposition Duplex 10 nM 10 nM 0.1 nM 0.1 nM relative to Name Avg SD Avg SDNM_000029.3 AD-60770.1 4.7 0.7 27.4 7.4 2292 AD-60771.1 4.6 1.2 10.9 5.92334 AD-60772.1 14.8 7.9 63.7 13.2 1594 AD-60773.1 43.9 8.7 88.8 5.41603 AD-60774.1 64.0 3.8 79.1 4.2 2399 AD-60775.1 8.4 2.8 62.2 8.8 843AD-60776.1 5.9 2.8 11.1 5.1 2309 AD-60777.1 4.3 1.7 26.7 5.3 2084AD-60778.1 11.5 7.4 52.5 15.5 1572 AD-60779.1 5.2 2.8 24.1 4.1 855AD-60780.1 5.7 2.3 12.0 5.2 2335 AD-60781.1 6.9 2.1 18.5 3.1 1894AD-60782.1 9.2 5.0 47.0 8.6 1737 AD-60783.1 15.9 9.0 35.7 7.6 2281AD-60784.1 5.6 1.0 12.3 2.5 2081 AD-60785.1 6.1 0.9 29.7 13.7 1741AD-60786.1 56.9 5.4 74.0 5.1 2386 AD-60787.1 94.4 0.7 104.4 6.0 912AD-60788.1 10.5 3.6 49.8 4.8 2286 AD-60789.1 5.2 2.0 25.3 16.0 2291AD-60790.1 70.4 3.3 86.3 19.7 2387 AD-60791.1 7.8 3.7 33.4 18.0 2329AD-60792.1 17.0 3.3 72.8 9.0 1574 AD-60793.1 18.4 5.6 58.3 12.5 2072AD-60794.1 54.3 11.5 101.6 4.3 1276 AD-60795.1 6.2 3.4 36.3 8.6 2270AD-60796.1 41.6 3.3 100.9 8.6 1568 AD-60797.1 72.5 2.8 97.1 6.1 857AD-60798.1 13.2 4.8 29.1 16.4 2316 AD-60799.1 29.7 7.5 86.2 4.2 1812AD-60800.1 31.0 7.5 81.8 11.2 1739 AD-60801.1 10.9 2.2 35.4 11.3 2328AD-60802.1 14.8 5.5 52.8 9.7 1742 AD-60803.1 29.2 10.9 71.4 9.6 830AD-60804.1 39.2 2.9 75.6 6.1 2400 AD-60805.1 60.3 10.1 97.4 2.3 2452AD-60806.1 89.3 11.8 92.8 6.5 880 AD-60807.1 64.1 3.7 91.5 10.1 913

TABLE 10 AGT IC₅₀ data in Hep3B Cells (21/23 mers) Duplex Name IC₅₀ (nM)AD-60771.1 0.028 AD-60776.1 0.033 AD-60780.1 0.038 AD-60784.1 0.045AD-60781.1 0.089

Example 2. In Vivo AGT Silencing—Amelioration of Preeclamptic Sequelae

Transgenic female Sprague-Dawley rats harboring the complete genomichuman AGT gene (e.g., [TGR(hAGT)L1623] (see, e.g., Bohlender J, et al.(1996) Hypertension 27: 535-540 and Bohlender J, et al. (2000) J Am SocNephrol 11: 2056-2061)) were surgically implemented with a device formeasuring blood pressure by telemetry. Subsequent to recovery from theprocedure, these rats were mated with transgenic male Sprague-Dawleyrats harboring the entire genomic human REN gene (e.g., [TGR(hREN)L10J](see, e.g., Bohlender J, et al. (1997) Hypertension 29: 428-434 andBohlender J, et al. (2000) J Am Soc Nephrol 11: 2056-2061)). The femaleprogeny of this cross (referred to herein as “PE rats”) are a model ofpreeclampsia and develop albuminuria and intrauterine growth restriction(IUGR), and have a blood pressure spike beginning around gestation day13 (see, e.g., FIG. 2A).

Beginning on day 3 of gestation, a subset of pregnant PE rats wasadministered 10 mg/kg siRNA targeting hAGT (AD-60771). A second subsetof pregnant PE rats were left untreated as a control. Dosing continuedevery third day to gestation day 15. Rats were sacrificed on about day19 of gestation, and blood and tissue samples were collected from themothers and the fetuses.

Maternal blood bressure was monitored throughout the experiment viasurgically implemented devices for measuring blood pressure bytelemetry. FIG. 2A shows that following administration of AD-60771,maternal mean arterial blood pressure was significantly lowered. Inaddition, as determined by ELISA analysis of serum albumin, maternalalbuminuria was significantly reduced following administration ofAD-60771 (FIG. 2B). These two conditions are the hallmarks ofpreeclampsia. The reduced blood pressure reduces cardiovascular eventsassociated with the disease, while reduced albuminuria is indicative ofimproved renal function.

Preeclampsia is also associated with reduced placental size, possiblyrelated to poor perfusion. As a result of the condition, fetal growth isimpaired. Following maternal administration of AD-60771, however,uteroplacental unit weight (FIG. 3A) and fetal weight (FIG. 3B) wereincreased, and there was normalization of the fetal brain:liver ratio(FIG. 3C), all indicative of a more normal placental and fetaldevelopment.

As determined by RT-qPCR analysis of mRNA expression of hAGT in maternalliver and placenta, it was shown that, while there is substantialsilencing of hAGT in the maternal liver (FIG. 4A), there was nosignificant silencing in the placenta (FIG. 4B). This indicates a lackof penetration of the iRNA into the placenta.

In addition, analysis of four maternal liver samples, four placentasamples, and eight fetal liver samples demonstrated that the exposure ofthe fetal liver to the iRNA was about 1.2 ng siRNA/g (below the limit ofdetection, 1.3 ng siRNA/g tissue) of tissue and that exposure of thematernal liver to the iRNA was about 265-fold greater than the exposureof the placenta to the iRNA (FIG. 5). Tissue exposure was determined bystem-loop qPCR, as previously described (see, e.g., Landesman, Y., etal. (2010) Silence 1:16.

Thus, maternal treatment with an iRNA targeting hAGT is superior tomaternal treatment with small-molecule renin-angiotensin inhibitors,such as ACE inhibitors, and angiotensin receptor blockers, as thesecompounds cross the placenta and are fetotoxic.

As the placenta acts as the conduit for nutrients to the fetus, andwaste removal from the fetus, its composition is critical. Accordingly,placental pathology was evaluated (i.e., by measuring the area of tissueslices immunohistochemically stained for cytokeratin). FIG. 6demonstrates that while the maternal portion of the placenta (themesometrial triangle; FIG. 6D) and the trophospongium (a uniform layerof cells which are precursors of differentiated trophoblasts; FIG. 6E)were unchanged by administration of AD-60771, overall placental size wasincreased (FIGS. 6B, 6C, and 6F and FIG. 6A for reference). Inparticular, the size of the labyrinth, which is the site of nutritionalexchange between fetal and maternal blood, was increased by maternaltreatment with AD-60771 (FIG. 6G).

The anti-angiogenic soluble fms-like tyrosine kinase-1 (sFLT1) and theangiogenic placental growth factor (PLGF), which are regulated byangiotensins, play a key role in preeclampsia, being released from theplacenta into the maternal blood stream and causing endothelialdysfunction. sFlt-1, is largely released from the placenta into thematernal blood stream and causes endothelial dysfunction. PLGF isplacental derived, and promotes angiogenesis. The ratio of sFlt-1:PLGFis diagnostic, with a higher sFlt-1:PLGF associated with more severedisease.

Evaluation of the mRNA expression of sFLT1 and PLGF by RT-qPCR analysisdemonstrated that the levels of both sFLT1 and PLGF were significantlyreduced in the maternal kidney (FIGS. 7A and 7B, respectively), butremained unchanged in the placenta (FIGS. 7C and 7D, respectively)following maternal administration of AD-60771. However, as sFlt-1 wasreduced to a greater extent than PLGF in the maternal kidney, the serumsFlt-1:PLGF ratio may be improved.

Agonistic autoantibodies to the angiotensin II receptor type I (ATI)have been identified in preeclamptic women. When pregnant rodents areexposed to AT1 autoantibodies, a preeclampsia-like syndrome develops. Asactivation of AT1 is associated with vasopressor effects as well asaldosterone secretion and sFlt-1 production, reduction of AT1autoantibodies would be expected to reduce the preeclamptic phenotype.Accordingly, the level of production of agonistic autoantibodies to theAT1 receptor (AT1-AA) may also be evaluated by biochromatography andsubsequent bioassay of isolated antibodies. AT1-AA levels were measuredas has been described (see, e.g., Dechend, et al. (2005) Hypertension45:742-746) by assessing the impact of isolated, purified AT1-AAantibodies on the spontaneous beating rate of neonatal ratcardiomyocytes. FIG. 8 demonstrates that there was a significantreduction in the level of AT1-AA following administration of AD-60771 topregnant PE rats.

Example 3. In Vivo AGT Silencing in a Transgenic Rat Model ofPreeclampsia

The AD-60771 duplex was tested for the ability to silence hAGTexpression in pregnant PE rats, described in the previous example.Beginning on day 3 of gestation, a subset of pregnant PE rats wasadministered 10 mg/kg siRNA targeting hAGT (AD-60771). Liver and bloodwere collected at gestation day 21 and the levels of hAGT and rAGTprotein and mRNA were assayed and compared to the levels of hAGT andrAGT protein and mRNA present in untreated, pregnant transgenic rats,pregnant Sprague-Dawley rats, and non-pregnant Sprague-Dawley rats. Theresults of these assays are shown in FIGS. 9A and 9B and demonstratethat there was an 80% reduction of circulating AT2 and a 95% reductionof circulating hAT1 in the pregnant PE rats treated with AD-60771 ascompared to control pregnant PE rats. A reduction of about 45% of ratAT2 was also observed in the pregnant PE rats treated with AD-60771 ascompared to control pregnant PE rats. A 95% silencing of hepatic AGTmRNA was observed in the pregnant PE rats treated with AD-60771 ascompared to control pregnant PE rats. FIGS. 9A and 9B also demonstratethat there was a significant reduction in the serum level of AT1 and AT2following administration of AD-60771 to pregnant PE rats.

TABLE 11Unmodified Sense and Antisense Strand Sequences of AGT dsRNAs (21/23 mers)Sense SEQ SEQ Duplex Oligo ID Antisense ID Location on Name NameSequence NO. Oligo Name Sequence NO. NM_000029.3 AD-67864.1 A-135936.1CGGGCAGCAGGGUCAGAAA 631 A-135937.1 UUUCUGACCCUGCUGCCCG 828   10-28AD-67865.1 A-135938.1 UCAGAAGUGGCCCCCGUGU 632 A-135939.1ACACGGGGGCCACUUCUGA 829   22-40_as AD-67866.1 A-135940.1CCCGUGUUGCCUAAGCAAA 633 A-135941.1 UUUGCUUAGGCAACACGGG 830  34-52_G19A_as AD-67867.1 A-135948.1 UGCACCUCCGGCCUGCAUA 634 A-135949.1UAUGCAGGCCGGAGGUGCA 831   76-94_G19A_as AD-67868.1 A-135950.1UGCAUGUCCCUGUGGCCUA 635 A-135951.1 UAGGCCACAGGGACAUGCA 832   89-107AD-67869.1 A-135952.1 UGUGGCCUCUUGGGGGUAA 636 A-135953.1UUACCCCCAAGAGGCCACA 833   99-117 AD-67870.1 A-135958.1GGUCAGAAGGCCUGGGUGA 637 A-135959.1 UCACCCAGGCCUUCUGACC 834  132-150_G19A_as AD-67871.1 A-135960.1 UGGGUGGUUGGCCUCAGGA 638 A-135961.1UCCUGAGGCCAACCACCCA 835  144-162 AD-67872.1 A-135962.1CCUCAGGCUGUCACACACA 639 A-135963.1 UGUGUGUGACAGCCUGAGG 836  155-173AD-67873.1 A-135964.1 CACACACCUAGGGAGAUGA 640 A-135965.1UCAUCUCCCUAGGUGUGUG 837  166-184 AD-67874.1 A-135966.1AGGGAGAUGCUCCCGUUUA 641 A-135967.1 UAAACGGGAGCAUCUCCCU 838  175-193AD-67875.1 A-135968.1 CCGUUUCUGGGAACCUUGA 642 A-135969.1UCAAGGUUCCCAGAAACGG 839  187- 205_G19A_as AD-67876.1 A-135970.1AACCUUGGCCCCGACUCCU 643 A-135971.1 AGGAGUCGGGGCCAAGGUU 840  198-216_asAD-67877.1 A-135972.1 CGACUCCUGCAAACUUCGA 644 A-135973.1UCGAAGUUUGCAGGAGUCG 841  209- 227_G19A_as AD-67878.1 A-135974.1AACUUCGGUAAAUGUGUAA 645 A-135975.1 UUACACAUUUACCGAAGUU 842  220-238_asAD-67879.1 A-135976.1 UGUGUAACUCGACCCUGCA 646 A-135977.1UGCAGGGUCGAGUUACACA 843  232-250_as AD-67880.1 A-135978.1ACCCUGCACCGGCUCACUA 647 A-135979.1 UAGUGAGCCGGUGCAGGGU 844  243-261AD-67881.1 A-135980.1 GCUCACUCUGUUCAGCAGU 648 A-135981.1ACUGCUGAACAGAGUGAGC 845  254-272_as AD-67882.1 A-135982.1UUCAGCAGUGAAACUCUGA 649 A-135983.1 UCAGAGUUUCACUGCUGAA 846  264-282AD-67883.1 A-135984.1 AAACUCUGCAUCGAUCACU 650 A-135985.1AGUGAUCGAUGCAGAGUUU 847  274-292_as AD-67884.1 A-135986.1CGAUCACUAAGACUUCCUA 651 A-135987.1 UAGGAAGUCUUAGUGAUCG 848  285-303_G19A_as AD-67885.1 A-135990.1 GAGGUCCCAGCGUGAGUGU 652 A-135991.1ACACUCACGCUGGGACCUC 849  307-325_as AD-67886.1 A-135994.1UUCUGGCAUCUGUCCUUCU 653 A-135995.1 AGAAGGACAGAUGCCAGAA 850  329-347_asAD-67887.1 A-135996.1 CCUUCUGGCCAGCCUGUGA 654 A-135997.1UCACAGGCUGGCCAGAAGG 851  342- 360_G19A_as AD-67888.1 A-135998.1AGCCUGUGGUCUGGCCAAA 655 A-135999.1 UUUGGCCAGACCACAGGCU 852  352-370_G19A_as AD-67889.1 A-136000.1 GGCCAAGUGAUGUAACCCU 656 A-136001.1AGGGUUACAUCACUUGGCC 853  364-382_as AD-67890.1 A-136002.1UGUAACCCUCCUCUCCAGA 657 A-136003.1 UCUGGAGAGGAGGGUUACA 854  374-392AD-67891.1 A-136004.1 UCUCCAGCCUGUGCACAGA 658 A-136005.1UCUGUGCACAGGCUGGAGA 855  385- 403_G19A_as AD-67892.1 A-136006.1UGCACAGGCAGCCUGGGAA 659 A-136007.1 UUCCCAGGCUGCCUGUGCA 856  396-414_asAD-67893.1 A-136008.1 CCUGGGAACAGCUCCAUCA 660 A-136009.1UGAUGGAGCUGUUCCCAGG 857  407-425 AD-67894.1 A-136010.1UCCAUCCCCACCCCUCAGA 661 A-136011.1 UCUGAGGGGUGGGGAUGGA 858  419-437AD-67895.1 A-136012.1 CCCUCAGCUAUAAAUAGGA 662 A-136013.1UCCUAUUUAUAGCUGAGGG 859  430- 448_G19A_as AD-67896.1 A-136014.1UAAAUAGGGCAUCGUGACA 663 A-136015.1 UGUCACGAUGCCCUAUUUA 860  440-458AD-67897.1 A-136016.1 AUCGUGACCCGGCCGGGGA 664 A-136017.1UCCCCGGCCGGGUCACGAU 861  450- 468_G19A_as AD-67898.1 A-136018.1CCGGGGGAAGAAGCUGCCA 665 A-136019.1 UGGCAGCUUCUUCCCCCGG 862  462-480_G19A_as AD-67899.1 A-136020.1 AGCUGCCGUUGUUCUGGGU 666 A-136021.1ACCCAGAACAACGGCAGCU 863  473-491_as AD-67900.1 A-136022.1UUCUGGGUACUACAGCAGA 667 A-136023.1 UCUGCUGUAGUACCCAGAA 864  484-502_asAD-67901.1 A-136024.1 UACAGCAGAAGGGUAUGCA 668 A-136025.1UGCAUACCCUUCUGCUGUA 865  494- 512_G19A_as AD-67902.1 A-136026.1UAUGCGGAAGCGAGCACCA 669 A-136027.1 UGGUGCUCGCUUCCGCAUA 866  507-525AD-67903.1 A-136028.1 CGAGCACCCCAGUCUGAGA 670 A-136029.1UCUCAGACUGGGGUGCUCG 867  517-535_as AD-67904.1 A-136032.1CUCCUGCCGGUGUGAGCCU 671 A-136033.1 AGGCUCACACCGGCAGGAG 868  539-557_asAD-67905.1 A-136034.1 UGUGAGCCUGAGGGCCACA 672 A-136035.1UGUGGCCCUCAGGCUCACA 869  549-567 AD-67906.1 A-136036.1GGGCCACCAUCCUCUGCCU 673 A-136037.1 AGGCAGAGGAUGGUGGCCC 870  560-578_asAD-67907.1 A-136038.1 CUGCCUCCUGGCCUGGGCU 674 A-136039.1AGCCCAGGCCAGGAGGCAG 871  573-591_as AD-67908.1 A-136042.1CCUGGCUGCAGGUGACCGA 675 A-136043.1 UCGGUCACCUGCAGCCAGG 872  594-612_G19A_as AD-67909.1 A-136044.1 UGACCGGGUGUACAUACAA 676 A-136045.1UUGUAUGUACACCCGGUCA 873  606-624 AD-67910.1 A-136046.1UACAUACACCCCUUCCACA 677 A-136047.1 UGUGGAAGGGGUGUAUGUA 874  616-634AD-67911.1 A-136048.1 UUCCACCUCGUCAUCCACA 678 A-136049.1UGUGGAUGACGAGGUGGAA 875  628-646_as AD-67912.1 A-136050.1UCAUCCACAAUGAGAGUAA 679 A-136051.1 UUACUCUCAUUGUGGAUGA 876  638-656AD-67913.1 A-136056.1 AAAGGCCAAUGCCGGGAAA 680 A-136057.1UUUCCCGGCAUUGGCCUUU 877  672- 690_G19A_as AD-67914.1 A-136058.1CCGGGAAGCCCAAAGACCA 681 A-136059.1 UGGUCUUUGGGCUUCCCGG 878  683-701AD-67915.1 A-136060.1 AAAGACCCCACCUUCAUAA 682 A-136061.1UUAUGAAGGUGGGGUCUUU 879  694-712 AD-67916.1 A-136062.1CCUUCAUACCUGCUCCAAU 683 A-136063.1 AUUGGAGCAGGUAUGAAGG 880  704-722_asAD-67917.1 A-136064.1 CUCCAAUUCAGGCCAAGAA 684 A-136065.1UUCUUGGCCUGAAUUGGAG 881  716-734 AD-67918.1 A-136066.1AGGCCAAGACAUCCCCUGU 685 A-136067.1 ACAGGGGAUGUCUUGGCCU 882  725-743_asAD-67919.1 A-136068.1 UCCCCUGUGGAUGAAAAGA 686 A-136069.1UCUUUUCAUCCACAGGGGA 883  736- 754_G19A_as AD-67920.1 A-136070.1AAAAGGCCCUACAGGACCA 687 A-136071.1 UGGUCCUGUAGGGCCUUUU 884  749-767_asAD-67921.1 A-136072.1 UACAGGACCAGCUGGUGCU 688 A-136073.1AGCACCAGCUGGUCCUGUA 885  758-776_as AD-67922.1 A-136078.1UUGACACCGAAGACAAGUU 689 A-136079.1 AACUUGUCUUCGGUGUCAA 886  791-809_asAD-67923.1 A-136080.1 ACAAGUUGAGGGCCGCAAU 690 A-136081.1AUUGCGGCCCUCAACUUGU 887  803-821_as AD-67924.1 A-136082.1GCCGCAAUGGUCGGGAUGA 691 A-136083.1 UCAUCCCGACCAUUGCGGC 888  814-832AD-67925.1 A-136084.1 CGGGAUGCUGGCCAACUUA 692 A-136085.1UAAGUUGGCCAGCAUCCCG 889  825-843 AD-67926.1 A-136086.1CAACUUCUUGGGCUUCCGU 693 A-136087.1 ACGGAAGCCCAAGAAGUUG 890  837-855_asAD-67927.1 A-136088.1 GGCUUCCGUAUAUAUGGCA 694 A-136089.1UGCCAUAUAUACGGAAGCC 891  847-865_as AD-67928.1 A-136090.1UAUGGCAUGCACAGUGAGA 695 A-136091.1 UCUCACUGUGCAUGCCAUA 892  859-877AD-67929.1 A-136092.1 ACAGUGAGCUAUGGGGCGU 696 A-136093.1ACGCCCCAUAGCUCACUGU 893  869-887_as AD-67930.1 A-136098.1CGUCCUCUCCCCAACGGCU 697 A-136099.1 AGCCGUUGGGGAGAGGACG 894  903-921_asAD-67931.1 A-136102.1 UUUGGCACCCUGGCCUCUA 698 A-136103.1UAGAGGCCAGGGUGCCAAA 895  925-943 AD-67932.1 A-136104.1CUGGCCUCUCUCUAUCUGA 699 A-136105.1 UCAGAUAGAGAGAGGCCAG 896  934-952_G19A_as AD-67933.1 A-136106.1 UAUCUGGGAGCCUUGGACA 700 A-136107.1UGUCCAAGGCUCCCAGAUA 897  946-964 AD-67934.1 A-136108.1UUGGACCACACAGCUGACA 701 A-136109.1 UGUCAGCUGUGUGGUCCAA 898  958-976_asAD-67935.1 A-136110.1 ACAGCUGACAGGCUACAGA 702 A-136111.1UCUGUAGCCUGUCAGCUGU 899  967- 985_G19A_as AD-67936.1 A-136112.1UACAGGCAAUCCUGGGUGU 703 A-136113.1 ACACCCAGGAUUGCCUGUA 900  980-998_asAD-67937.1 A-136114.1 UCCUGGGUGUUCCUUGGAA 704 A-136115.1UUCCAAGGAACACCCAGGA 901  989-1007_as AD-67938.1 A-136116.1UUGGAAGGACAAGAACUGA 705 A-136117.1 UCAGUUCUUGUCCUUCCAA 902 1002-1020AD-67939.1 A-136118.1 AGAACUGCACCUCCCGGCU 706 A-136119.1AGCCGGGAGGUGCAGUUCU 903 1013-1031_as AD-67940.1 A-136122.1UGCGCACAAGGUCCUGUCU 707 A-136123.1 AGACAGGACCUUGUGCGCA 904 1035-1053_asAD-67947.1 A-136126.1 CUGCAGGCUGUACAGGGCA 708 A-136127.1UGCCCUGUACAGCCUGCAG 905 1057-1075 AD-67948.1 A-136128.1UACAGGGCCUGCUAGUGGA 709 A-136129.1 UCCACUAGCAGGCCCUGUA 906 1067-1085AD-67949.1 A-136130.1 UAGUGGCCCAGGGCAGGGA 710 A-136131.1UCCCUGCCCUGGGCCACUA 907 1079-1097 AD-67950.1 A-136132.1AGGGCAGGGCUGAUAGCCA 711 A-136133.1 UGGCUAUCAGCCCUGCCCU 908 1088-1106_asAD-67951.1 A-136134.1 UAGCCAGGCCCAGCUGCUA 712 A-136135.1UAGCAGCUGGGCCUGGCUA 909 1101- 1119_G19A_as AD-67952.1 A-136136.1AGCUGCUGCUGUCCACGGU 713 A-136137.1 ACCGUGGACAGCAGCAGCU 910 1112-1130_asAD-67954.1 A-136140.1 UGGGCGUGUUCACAGCCCA 714 A-136141.1UGGGCUGUGAACACGCCCA 911 1133-1151 AD-67955.1 A-136142.1CACAGCCCCAGGCCUGCAA 715 A-136143.1 UUGCAGGCCUGGGGCUGUG 912 1143-1161AD-67956.1 A-136144.1 CUGCACCUGAAGCAGCCGU 716 A-136145.1ACGGCUGCUUCAGGUGCAG 913 1156-1174_as AD-67957.1 A-136146.1AGCAGCCGUUUGUGCAGGA 717 A-136147.1 UCCUGCACAAACGGCUGCU 914 1166-1184_G19A_as AD-67958.1 A-136148.1 UGCAGGGCCUGGCUCUCUA 718 A-136149.1UAGAGAGCCAGGCCCUGCA 915 1178-1196_as AD-67959.1 A-136150.1UGGCUCUCUAUACCCCUGU 719 A-136151.1 ACAGGGGUAUAGAGAGCCA 916 1187-1205_asAD-67960.1 A-136152.1 ACCCCUGUGGUCCUCCCAA 720 A-136153.1UUGGGAGGACCACAGGGGU 917 1198-1216 AD-67962.1 A-136156.1CUGGACUUCACAGAACUGA 721 A-136157.1 UCAGUUCUGUGAAGUCCAG 918 1222-1240_G19A_as AD-67963.1 A-136158.1 AGAACUGGAUGUUGCUGCU 722 A-136159.1AGCAGCAACAUCCAGUUCU 919 1233-1251_as AD-67964.1 A-136160.1UUGCUGCUGAGAAGAUUGA 723 A-136161.1 UCAAUCUUCUCAGCAGCAA 920 1244-1262_asAD-67965.1 A-136162.1 AGAAGAUUGACAGGUUCAU 724 A-136163.1AUGAACCUGUCAAUCUUCU 921 1253-1271_as AD-67966.1 A-136164.1AGGUUCAUGCAGGCUGUGA 725 A-136165.1 UCACAGCCUGCAUGAACCU 922 1264-1282_asAD-67967.1 A-136166.1 GCUGUGACAGGAUGGAAGA 726 A-136167.1UCUUCCAUCCUGUCACAGC 923 1276-1294_as AD-67968.1 A-136168.1UGGAAGACUGGCUGCUCCA 727 A-136169.1 UGGAGCAGCCAGUCUUCCA 924 1288-1306AD-67970.1 A-136172.1 AUGGGAGCCAGUGUGGACA 728 A-136173.1UGUCCACACUGGCUCCCAU 925 1309-1327_as AD-67971.1 A-136174.1UGUGGACAGCACCCUGGCU 729 A-136175.1 AGCCAGGGUGCUGUCCACA 926 1320-1338_asAD-67972.1 A-136176.1 CCUGGCUUUCAACACCUAA 730 A-136177.1UUAGGUGUUGAAAGCCAGG 927 1332-1350 AD-67973.1 A-136178.1CAACACCUACGUCCACUUA 731 A-136179.1 UAAGUGGACGUAGGUGUUG 928 1341-1359AD-67974.1 A-136180.1 CACUUCCAAGGGAAGAUGA 732 A-136181.1UCAUCUUCCCUUGGAAGUG 929 1354-1372_as AD-67975.1 A-136182.1GGGAAGAUGAAGGGCUUCU 733 A-136183.1 AGAAGCCCUUCAUCUUCCC 930 1363-1381_asAD-67976.1 A-136184.1 GCUUCUCCCUGCUGGCCGA 734 A-136185.1UCGGCCAGCAGGGAGAAGC 931 1376-1394_as AD-67978.1 A-136188.1CCAGGAGUUCUGGGUGGAA 735 A-136189.1 UUCCACCCAGAACUCCUGG 932 1398-1416AD-67979.1 A-136190.1 UGGGUGGACAACAGCACCU 736 A-136191.1AGGUGCUGUUGUCCACCCA 933 1408-1426_as AD-67980.1 A-136192.1ACAGCACCUCAGUGUCUGU 737 A-136193.1 ACAGACACUGAGGUGCUGU 934 1418-1436_asAD-67981.1 A-136194.1 UGUCUGUUCCCAUGCUCUA 738 A-136195.1UAGAGCAUGGGAACAGACA 935 1430-1448 AD-67982.1 A-136196.1CAUGCUCUCUGGCAUGGGA 739 A-136197.1 UCCCAUGCCAGAGAGCAUG 936 1440-1458AD-67983.1 A-136198.1 AUGGGCACCUUCCAGCACU 740 A-136199.1AGUGCUGGAAGGUGCCCAU 937 1453-1471_as AD-67984.1 A-136200.1UUCCAGCACUGGAGUGACA 741 A-136201.1 UGUCACUCCAGUGCUGGAA 938 1462-1480_asAD-67986.1 A-136204.1 AGGACAACUUCUCGGUGAA 742 A-136205.1UUCACCGAGAAGUUGUCCU 939 1484-1502 AD-67987.1 A-136206.1UCGGUGACUCAAGUGCCCU 743 A-136207.1 AGGGCACUUGAGUCACCGA 940 1495-1513_asAD-67988.1 A-136208.1 UGCCCUUCACUGAGAGCGA 744 A-136209.1UCGCUCUCAGUGAAGGGCA 941 1508-1526 AD-67989.1 A-136210.1UGAGAGCGCCUGCCUGCUA 745 A-136211.1 UAGCAGGCAGGCGCUCUCA 942 1518-1536_G19A_as AD-67990.1 A-136212.1 CCUGCUGCUGAUCCAGCCU 746 A-136213.1AGGCUGGAUCAGCAGCAGG 943 1530-1548_as AD-67991.1 A-136214.1AUCCAGCCUCACUAUGCCU 747 A-136215.1 AGGCAUAGUGAGGCUGGAU 944 1540-1558_asAD-67992.1 A-136216.1 UAUGCCUCUGACCUGGACA 748 A-136217.1UGUCCAGGUCAGAGGCAUA 945 1552-1570_as AD-67993.1 A-136218.1ACCUGGACAAGGUGGAGGA 749 A-136219.1 UCCUCCACCUUGUCCAGGU 946 1562-1580_G19A_as AD-67994.1 A-136220.1 UGGAGGGUCUCACUUUCCA 750 A-136221.1UGGAAAGUGAGACCCUCCA 947 1574-1592_as AD-67995.1 A-136222.1CACUUUCCAGCAAAACUCA 751 A-136223.1 UGAGUUUUGCUGGAAAGUG 948 1584-1602AD-67996.1 A-136224.1 AAAACUCCCUCAACUGGAU 752 A-136225.1AUCCAGUUGAGGGAGUUUU 949 1595-1613_as AD-67997.1 A-136226.1AACUGGAUGAAGAAACUAU 753 A-136227.1 AUAGUUUCUUCAUCCAGUU 950 1606-1624_asAD-67998.1 A-136228.1 AAACUAUCUCCCCGGACCA 754 A-136229.1UGGUCCGGGGAGAUAGUUU 951 1618-1636_as AD-67999.1 A-136230.1CCGGACCAUCCACCUGACA 755 A-136231.1 UGUCAGGUGGAUGGUCCGG 952 1629-1647AD-68001.1 A-136234.1 UGCCCCAACUGGUGCUGCA 756 A-136235.1UGCAGCACCAGUUGGGGCA 953 1649-1667_as AD-68002.1 A-136236.1UGCUGCAAGGAUCUUAUGA 757 A-136237.1 UCAUAAGAUCCUUGCAGCA 954 1661-1679_asAD-68003.1 A-136238.1 UCUUAUGACCUGCAGGACA 758 A-136239.1UGUCCUGCAGGUCAUAAGA 955 1672-1690 AD-68004.1 A-136240.1UGCAGGACCUGCUCGCCCA 759 A-136241.1 UGGGCGAGCAGGUCCUGCA 956 1682-1700_asAD-68005.1 A-136242.1 UCGCCCAGGCUGAGCUGCA 760 A-136243.1UGCAGCUCAGCCUGGGCGA 957 1694-1712 AD-68006.1 A-136244.1UGAGCUGCCCGCCAUUCUA 761 A-136245.1 UAGAAUGGCGGGCAGCUCA 958 1704-1722_G19A_as AD-68009.1 A-136250.1 UGCAAAAAUUGAGCAAUGA 762 A-136251.1UCAUUGCUCAAUUUUUGCA 959 1739-1757_as AD-68010.1 A-136252.1AGCAAUGACCGCAUCAGGA 763 A-136253.1 UCCUGAUGCGGUCAUUGCU 960 1750-1768_G19A_as AD-68011.1 A-136254.1 CAUCAGGGUGGGGGAGGUA 764 A-136255.1UACCUCCCCCACCCUGAUG 961 1761- 1779_G19A_as AD-68012.1 A-136256.1GGGGAGGUGCUGAACAGCA 765 A-136257.1 UGCUGUUCAGCACCUCCCC 962 1771-1789_asAD-68013.1 A-136258.1 AACAGCAUUUUUUUUGAGA 766 A-136259.1UCUCAAAAAAAAUGCUGUU 963 1783-1801 AD-68014.1 A-136260.1UUUUUGAGCUUGAAGCGGA 767 A-136261.1 UCCGCUUCAAGCUCAAAAA 964 1793-1811_asAD-68017.1 A-136266.1 AGAGUCUACCCAACAGCUU 768 A-136267.1AAGCUGUUGGGUAGACUCU 965 1827-1845_as AD-68018.1 A-136268.1CAACAGCUUAACAAGCCUA 769 A-136269.1 UAGGCUUGUUAAGCUGUUG 966 1837-1855_G19A_as AD-68019.1 A-136270.1 CAAGCCUGAGGUCUUGGAA 770 A-136271.1UUCCAAGACCUCAGGCUUG 967 1848- 1866_G19A_as AD-68020.1 A-136272.1CUUGGAGGUGACCCUGAAA 771 A-136273.1 UUUCAGGGUCACCUCCAAG 968 1860-1878AD-68021.1 A-136274.1 ACCCUGAACCGCCCAUUCA 772 A-136275.1UGAAUGGGCGGUUCAGGGU 969 1870-1888 AD-68022.1 A-136276.1GCCCAUUCCUGUUUGCUGU 773 A-136277.1 ACAGCAAACAGGAAUGGGC 970 1880-1898_asAD-68025.1 A-136284.1 CACUUCCUGGGCCGCGUGA 774 A-136285.1UCACGCGGCCCAGGAAGUG 971 1924- 1942_G19A_as AD-68026.1 A-136286.1CGCGUGGCCAACCCGCUGA 775 A-136287.1 UCAGCGGGUUGGCCACGCG 972 1936-1954_asAD-68027.1 A-136288.1 ACCCGCUGAGCACAGCAUA 776 A-136289.1UAUGCUGUGCUCAGCGGGU 973 1946- 1964_G19A_as AD-68028.1 A-136290.1ACAGCAUGAGGCCAGGGCA 777 A-136291.1 UGCCCUGGCCUCAUGCUGU 974 1957-1975AD-68029.1 A-136292.1 CAGGGCCCCAGAACACAGU 778 A-136293.1ACUGUGUUCUGGGGCCCUG 975 1969-1987_as AD-68032.1 A-136298.1UCUGCCCCUGGCCUUUGAA 779 A-136299.1 UUCAAAGGCCAGGGGCAGA 976 2001-2019_G19A_as AD-68033.1 A-136300.1 UUUGAGGCAAAGGCCAGCA 780 A-136301.1UGCUGGCCUUUGCCUCAAA 977 2014-2032_as AD-68034.1 A-136302.1AGGCCAGCAGCAGAUAACA 781 A-136303.1 UGUUAUCUGCUGCUGGCCU 978 2024-2042_asAD-68035.1 A-136304.1 AGAUAACAACCCCGGACAA 782 A-136305.1UUGUCCGGGGUUGUUAUCU 979 2035-2053_as AD-68036.1 A-136306.1CCGGACAAAUCAGCGAUGU 783 A-136307.1 ACAUCGCUGAUUUGUCCGG 980 2046-2064_asAD-68037.1 A-136308.1 AGCGAUGUGUCACCCCCAA 784 A-136309.1UUGGGGGUGACACAUCGCU 981 2057- 2075_G19A_as AD-68084.2 A-136314.1UUCUAAUGAGUCGACUUUA 785 A-136315.1 UAAAGUCGACUCAUUAGAA 982 2090-2108_G19A_as AD-68085.2 A-136320.1 GUUUCUCCUUGGUCUAAGU 786 A-136321.1ACUUAGACCAAGGAGAAAC 983 2124-2142_as AD-68086.2 A-136328.1AGCCUGCAGCGGCACAAAU 787 A-136329.1 AUUUGUGCCGCUGCAGGCU 984 2167-2185_asAD-68087.2 A-136330.1 CACAAAUGCACCUCCCAGU 788 A-136331.1ACUGGGAGGUGCAUUUGUG 985 2179-2197_as AD-68088.2 A-136332.1ACCUCCCAGUUUGCUGGGU 789 A-136333.1 ACCCAGCAAACUGGGAGGU 986 2188-2206_asAD-68089.2 A-136334.1 UGCUGGGUUUAUUUUAGAA 790 A-136335.1UUCUAAAAUAAACCCAGCA 987 2199- 2217_G19A_as AD-68090.2 A-136340.1CAAGAACCAGUGUUUAGCA 791 A-136341.1 UGCUAAACACUGGUUCUUG 988 2234-2252_G19A_as AD-68091.2 A-136354.1 AGUGUUCCCUUUUCAAGUU 792 A-136355.1AACUUGAAAAGGGAACACU 989 2309-2327_as AD-68092.2 A-136356.1UUCAAGUUGAGAACAAAAA 793 A-136357.1 UUUUUGUUCUCAACUUGAA 990 2320-2338_asAD-68093.2 A-136358.1 CAAAAAUUGGGUUUUAAAA 794 A-136359.1UUUUAAAACCCAAUUUUUG 991 2333-2351_as AD-68094.2 A-136362.1AAAGUAUACAUUUUUGCAU 795 A-136363.1 AUGCAAAAAUGUAUACUUU 992 2354-2372_asAD-68095.2 A-136368.1 UUUAGUGUCUUGAAUGUAA 796 A-136369.1UUACAUUCAAGACACUAAA 993 2388-2406_as AD-68096.2 A-136370.1GAAUGUAAGAACAUGACCU 797 A-136371.1 AGGUCAUGUUCUUACAUUC 994 2399-2417_asAD-68097.2 A-136374.1 UGUAGUGUCUGUAAUACCU 798 A-136375.1AGGUAUUACAGACACUACA 995 2421-2439_as AD-68098.2 A-136376.1UGUAAUACCUUAGUUUUUU 799 A-136377.1 AAAAAACUAAGGUAUUACA 996 2430-2448_asAD-68099.2 A-136378.1 UUUUUUCCACAGAUGCUUA 800 A-136379.1UAAGCAUCUGUGGAAAAAA 997 2443- 2461_G19A_as AD-68100.2 A-136382.1UUUUUGAACAAUACGUGAA 801 A-136383.1 UUCACGUAUUGUUCAAAAA 998 2465-2483_asAD-68101.2 A-136392.1 ACCAUAGCUGGUUAUUUCU 802 A-136393.1AGAAAUAACCAGCUAUGGU 999 2519-2537_as AD-68102.2 A-136394.1UUAUUUCUCCCUUGUGUUA 803 A-136395.1 UAACACAAGGGAGAAAUAA 1000 2530-2548_asAD-68116.1 A-136312.1 UCCCACCUUUUCUUCUAAU 804 A-136313.1AUUAGAAGAAAAGGUGGGA 1001 2078-2096_as AD-68117.1 A-136316.1UCGACUUUGAGCUGGAAAG 805 A-136317.1 CUUUCCAGCUCAAAGUCGA 1002 2100-2118_asAD-68118.1 A-136318.1 CUGGAAAGCAGCCGUUUCU 806 A-136319.1AGAAACGGCUGCUUUCCAG 1003 2111-2129_as AD-68119.1 A-136322.1UGGUCUAAGUGUGCUGCAU 807 A-136323.1 AUGCAGCACACUUAGACCA 1004 2133-2151_asAD-68120.1 A-136324.1 GCUGCAUGGAGUGAGCAGU 808 A-136325.1ACUGCUCACUCCAUGCAGC 1005 2145-2163_as AD-68121.1 A-136326.1UGAGCAGUAGAAGCCUGCA 809 A-136327.1 UGCAGGCUUCUACUGCUCA 1006 2156-2174_asAD-68122.1 A-136336.1 UUAGAGAAUGGGGGUGGGA 810 A-136337.1UCCCACCCCCAUUCUCUAA 1007 2212- 2230_G19A_as AD-68123.1 A-136338.1GGGUGGGGAGGCAAGAACA 811 A-136339.1 UGUUCUUGCCUCCCCACCC 1008 2223-2241AD-68124.1 A-136342.1 UGUUUAGCGCGGGACUACU 812 A-136343.1AGUAGUCCCGCGCUAAACA 1009 2244-2262_as AD-68125.1 A-136344.1GGACUACUGUUCCAAAAAG 813 A-136345.1 CUUUUUGGAACAGUAGUCC 1010 2255-2273_asAD-68126.1 A-136350.1 AGCUUGUUUGUGAAACAAA 814 A-136351.1UUUGUUUCACAAACAAGCU 1011 2288-2306_as AD-68127.1 A-136352.1AAACAAAAAAGUGUUCCCU 815 A-136353.1 AGGGAACACUUUUUUGUUU 1012 2300-2318_asAD-68128.1 A-136360.1 UUUUAAAAUUAAAGUAUAA 816 A-136361.1UUAUACUUUAAUUUUAAAA 1013 2344-2362 AD-68129.1 A-136364.1UUUUGCAUUGCCUUCGGUU 817 A-136365.1 AACCGAAGGCAAUGCAAAA 1014 2365-2383_asAD-68130.1 A-136366.1 UUCGGUUUGUAUUUAGUGU 818 A-136367.1ACACUAAAUACAAACCGAA 1015 2377-2395_as AD-68131.1 A-136372.1AACAUGACCUCCGUGUAGU 819 A-136373.1 ACUACACGGAGGUCAUGUU 1016 2408-2426_asAD-68132.1 A-136380.1 CAGAUGCUUGUGAUUUUUA 820 A-136381.1UAAAAAUCACAAGCAUCUG 1017 2452- 2470_G19A_as AD-68133.1 A-136384.1UACGUGAAAGAUGCAAGCA 821 A-136385.1 UGCUUGCAUCUUUCACGUA 1018 2476-2494_asAD-68134.1 A-136386.1 UGCAAGCACCUGAAUUUCU 822 A-136387.1AGAAAUUCAGGUGCUUGCA 1019 2487-2505_as AD-68135.1 A-136388.1GAAUUUCUGUUUGAAUGCA 823 A-136389.1 UGCAUUCAAACAGAAAUUC 1020 2498-2516_G19A_as AD-68136.1 A-136390.1 UUUGAAUGCGGAACCAUAA 824 A-136391.1UUAUGGUUCCGCAUUCAAA 1021 2507- 2525_G19A_as AD-68137.1 A-136396.1UUGUGUUAGUAAUAAACGU 825 A-136397.1 ACGUUUAUUACUAACACAA 1022 2541-2559_asAD-68138.1 A-136398.1 AUAAACGUCUUGCCACAAU 826 A-136399.1AUUGUGGCAAGACGUUUAU 1023 2552-2570_as AD-68139.1 A-136400.1UGCCACAAUAAGCCUCCAA 827 A-136401.1 UUGGAGGCUUAUUGUGGCA 1024 2562-2580_as

TABLE 12 AGT single dose screen in Hep3B cells (21/23 mers) 10 nM 10 nM0.1 nM 0.1 nM Duplex Name Avg SD Avg SD AD-67864.1 86.08 0.42 100.664.93 AD-67865.1 90.98 0.44 97.56 4.30 AD-67866.1 107.85 3.17 108.20 1.59AD-67867.1 96.24 5.18 96.22 4.24 AD-67868.1 92.25 0.45 93.87 0.92AD-67869.1 107.24 8.40 96.54 3.78 AD-67870.1 89.43 1.75 96.84 0.47AD-67871.1 100.66 4.93 96.91 5.22 AD-67872.1 92.27 2.26 87.75 7.73AD-67873.1 118.49 6.38 105.63 3.10 AD-67874.1 98.36 8.18 99.57 1.46AD-67875.1 98.55 2.89 100.76 14.27 AD-67876.1 98.951 5.33 105.24 0.51AD-67877.1 104.94 5.14 100.97 2.47 AD-67878.1 101.79 7.47 100.61 0.98AD-67879.1 99.66 6.34 101.66 0.49 AD-67880.1 99.23 1.94 93.79 9.63AD-67881.1 95.84 0.93 104.16 2.04 AD-67882.1 97.52 1.43 98.09 9.60AD-67883.1 94.53 1.85 95.88 3.75 AD-67884.1 110.16 5.39 105.25 1.54AD-67885.1 101.70 4.48 97.94 5.75 AD-67886.1 107.09 2.09 102.03 3.00AD-67887.1 95.51 0.46 94.24 4.15 AD-67888.1 105.98 1.55 101.03 5.44AD-67889.1 89.42 0.87 102.44 5.52 AD-67890.1 96.51 0.94 99.34 6.81AD-67891.1 89.73 0.43 102.01 0.99 AD-67892.1 85.81 3.36 91.97 3.60AD-67893.1 104.89 2.05 93.25 3.65 AD-67894.1 95.95 6.57 100.61 0.98AD-67895.1 87.61 3.43 92.99 5.92 AD-67896.1 102.36 0.50 102.37 1.50AD-67897.1 89.23 6.55 101.66 1.49 AD-67898.1 87.68 6.01 97.89 3.83AD-67899.1 10.98 1.28 28.08 7.87 AD-67900.1 16.91 2.47 42.49 5.81AD-67901.1 51.36 1.00 73.53 10.77 AD-67902.1 32.91 6.25 49.54 4.60AD-67903.1 8.23 3.73 25.89 8.71 AD-67904.1 76.02 3.35 92.35 5.88AD-67905.1 99.22 0.97 103.21 7.58 AD-67906.1 12.68 6.52 31.29 0.76AD-67907.1 25.24 0.86 68.71 0.67 AD-67908.1 15.32 6.82 41.71 0.40AD-67909.1 57.21 2.52 81.72 1.60 AD-67910.1 11.66 3.10 54.68 6.68AD-67911.1 40.57 0.39 60.72 4.16 AD-67912.1 21.45 0.84 50.16 2.70AD-67913.1 44.86 5.26 69.55 5.78 AD-67914.1 13.41 4.82 14.58 2.84AD-67915.1 38.73 3.41 62.81 7.67 AD-67916.1 5.21 2.70 19.28 6.84AD-67917.1 16.92 1.57 50.03 3.91 AD-67918.1 52.71 4.38 95.29 6.53AD-67919.1 86.98 0.85 96.94 6.17 AD-67920.1 28.31 8.20 62.46 8.84AD-67921.1 40.44 1.38 65.06 20.38 AD-67922.1 37.99 0.93 68.09 5.00AD-67923.1 9.52 0.97 31.95 1.09 AD-67924.1 20.28 2.28 53.38 6.78AD-67925.1 15.78 1.46 35.24 4.82 AD-67926.1 12.85 0.88 28.20 0.41AD-67927.1 39.78 2.72 70.72 17.16 AD-67928.1 48.92 8.11 76.56 4.12AD-67929.1 95.53 3.27 103.52 6.08 AD-67930.1 7.70 3.25 38.13 1.12AD-67931.1 62.59 2.14 83.32 7.74 AD-67932.1 12.85 4.97 48.21 4.95AD-67933.1 75.51 4.06 99.92 1.95 AD-67934.1 95.55 4.21 95.85 1.87AD-67935.1 6.92 0.06 26.43 22.43 AD-67936.1 30.62 3.29 64.66 5.06AD-67937.1 11.89 1.33 46.23 25.91 AD-67938.1 84.08 4.94 94.19 0.46AD-67939.1 49.09 0.24 76.31 13.39 AD-67940.1 77.85 0.76 90.12 5.29AD-67947.1 81.74 5.60 89.68 3.07 AD-67948.1 57.40 3.93 75.94 12.96AD-67949.1 89.04 1.30 98.84 4.35 AD-67950.1 30.38 6.93 50.01 4.40AD-67951.1 57.60 12.32 84.26 2.89 AD-67952.1 30.08 9.84 71.83 1.76AD-67954.1 63.41 2.17 83.16 5.29 AD-67955.1 66.78 1.30 88.74 2.60AD-67956.1 48.82 4.54 84.38 7.02 AD-67957.1 66.79 1.96 96.46 3.78AD-67958.1 11.88 6.31 28.49 7.58 AD-67959.1 50.08 5.87 78.05 1.14AD-67960.1 53.33 9.104 72.41 9.90 AD-67962.1 17.29 5.41 54.30 13.17AD-67963.1 19.63 2.11 49.27 15.43 AD-67964.1 14.83 0.14 46.79 6.40AD-67965.1 15.94 9.26 36.16 16.09 AD-67966.1 41.84 9.95 80.28 3.54AD-67967.1 20.10 2.94 45.85 6.71 AD-67968.1 24.10 0.47 45.77 0.22AD-67970.1 41.61 6.09 59.36 1.16 AD-67971.1 92.20 3.16 97.48 4.29AD-67972.1 24.48 3.46 60.84 2.68 AD-67973.1 67.49 2.31 93.19 4.56AD-67974.1 22.421 0.76 62.87 17.33 AD-67975.1 20.05 2.157 52.74 5.93AD-67976.1 19.21 5.30 54.84 6.96 AD-67978.1 17.36 1.10 60.49 14.96AD-67979.1 104.18 7.14 104.08 3.06 AD-67980.1 17.85 4.84 43.65 9.34AD-67981.1 28.91 4.09 63.61 0.62 AD-67982.1 91.54 0.44 103.34 AD-67983.176.45 1.87 97.09 0.95 AD-67984.1 23.20 0.34 75.39 8.84 AD-67986.1 10.162.32 43.00 0.21 AD-67987.1 82.21 0.80 90.90 10.66 AD-67988.1 22.56 7.8059.12 9.52 AD-67989.1 34.16 10.22 67.14 9.83 AD-67990.1 24.33 3.68 36.353.20 AD-67991.1 81.13 3.97 100.55 3.94 AD-67992.1 56.20 3.30 97.78 1.91AD-67993.1 29.38 5.01 78.86 13.08 AD-67994.1 20.30 4.44 30.93 AD-67995.113.97 2.72 37.27 5.82 AD-67996.1 14.48 0.49 30.62 6.25 AD-67997.1 35.607.78 70.86 12.09 AD-67998.1 29.38 5.01 62.37 15.13 AD-67999.1 58.52 6.5886.27 9.70 AD-68001.1 39.90 11.75 68.76 5.38 AD-68002.1 70.36 2.41 87.501.61 AD-68003.1 31.65 2.78 86.01 2.10 AD-68004.1 23.47 1.60 42.71 1.04AD-68005.1 30.41 6.21 68.42 1.00 AD-68006.1 20.44 8.64 41.45 5.87AD-68009.1 26.59 6.45 50.87 12.58 AD-68010.1 18.01 2.98 41.65 11.68AD-68011.1 48.64 4.28 75.01 10.99 AD-68012.1 21.28 0.41 66.82 13.98AD-68013.1 101.46 9.93 109.28 4.28 AD-68014.1 43.62 7.44 71.82 0.35AD-68017.1 16.05 11.61 38.14 14.23 AD-68018.1 14.74 0.57 62.02 9.38AD-68019.1 20.85 3.66 84.24 1.23 AD-68020.1 31.12 4.86 70.58 16.45AD-68021.1 18.98 3.8 56.30 10.96 AD-68022.1 16.41 9.93 27.87 15.96AD-68025.1 86.91 1.70 99.51 3.41 AD-68026.1 26.43 9.50 73.34 1.79AD-68027.1 39.43 0.38 72.57 1.42 AD-68028.1 56.98 3.34 80.52 AD-68029.149.97 3.42 85.43 2.93 AD-68032.1 71.12 3.48 91.96 6.30 AD-68033.1 102.292.50 99.13 0.97 AD-68034.1 15.54 3.32 32.03 3.91 AD-68035.1 19.40 6.8849.77 6.32 AD-68036.1 16.23 8.62 29.22 4.28 AD-68037.1 20.40 5.24 49.319.13 AD-68084.2 15.14 3.24 42.67 5.63 AD-68085.2 17.11 5.52 30.76 11.20AD-68086.2 8.96 0 21.05 2.98 AD-68087.2 22.54 14.44 26.79 37.89AD-68088.2 34.54 2.19 76.58 1.87 AD-68089.2 27.02 5.91 59.45 13.85AD-68090.2 19.90 0.97 35.94 7.00 AD-68091.2 15.18 0.74 32.19 3.77AD-68092.2 13.21 0.25 40.48 1.38 AD-68093.2 13.94 3.11 52.91 25.17AD-68094.2 48.22 7.06 89.18 25.43 AD-68095.2 83.02 5.69 81.51 1.19AD-68096.2 74.34 5.82 75.78 1.21 AD-68097.2 88.32 4.32 86.30 7.18AD-68098.2 69.49 0.34 69.49 1.02 AD-68099.2 95.96 3.76 51.05 72.19AD-68100.2 81.51 1.19 92.05 3.60 AD-68101.2 76.34 2.99 74.83 5.13AD-68102.2 71.02 4.52 76.99 6.78 AD-68116.1 9.94 0.38 31.41 3.53AD-68117.1 18.24 0.62 51.01 7.96 AD-68118.1 16.61 0.32 31.54 7.50AD-68119.1 48.03 3.76 77.61 12.49 AD-68120.1 20.17 11.80 72.58 10.63AD-68121.1 16.25 4.55 57.64 25.65 AD-68122.1 57.36 5.33 97.32 4.76AD-68123.1 15.84 1.24 45.55 2.00 AD-68124.1 24.62 8.40 51.57 19.72AD-68125.1 18.71 9.38 35.98 6.14 AD-68126.1 13.13 6.42 47.97 1.41AD-68127.1 41.04 12.66 85.58 2.93 AD-68128.1 62.20 16.27 107.38 9.46AD-68129.1 79.00 81.25 3.18 AD-68130.1 72.97 2.50 88.00 3.88 AD-68131.1134.88 9.24 92.09 5.41 AD-68132.1 81.05 5.95 89.85 3.96 AD-68133.1 85.282.50 104.25 2.04 AD-68134.1 62.63 0.92 83.94 6.98 AD-68135.1 72.95 0.3579.05 3.87 AD-68136.1 74.86 10.60 88.88 AD-68137.1 85.08 6.24 90.50 4.87AD-68138.1 76.05 1.11 76.34 2.99 AD-68139.1 102.25 8.01 99.42 6.81

Example 4: In Vivo AGT Silencing in a Mouse Expressing hAGT

A series of experiments were performed to assess hAGT target knockdownin mice expressing human AGT (hAGT) from an AAV8 expression vector whichhas strong liver tropism. Briefly, mice were infected with 1×10¹¹ AAV8particles containing an expression construct for hAGT under the controlof a constitutive promoter. Two weeks after infection, mice wereadministered a single 3.0 mg/kg, 1.0 mg/kg, 0.3 mg/kg, or 0.1 mg/kg doseof an siRNA targeting hAGT (AD-67327.2 or AD-67335.2), or PBS as acontrol (n=4 per group). The nucleotide sequences of the agents assessedin these experiments are provided in Table 13.

TABLE 13Modified Sense and Antisense Strand Sequences of AGT dsRNAs (21/23 mers)Sense Strand/ SEQ Duplex Location on Antisense Sense Sequence/AntisenseID Parent Name NM_000029.3 Strand Sequence NO. Duplex AD-67327 2081-2101A-134527 uscsucccAfcCfUfUfuucuucuaauL96 1025 AD-60784 2079-2101 A-134528asUfsuagAfagaaaagGfuGfggagascsu 1026 AD-67335 1894-1914 A-134536usgsuuugCfuGfUfGfuaugaucaaaL96 1027 AD-60781 1892-1914 A-134538usUfsugaUfcAfUfacacAfgCfaaacasgsg 1028

At days 0, 3, 7, and 14, after administration of the agents, bloodsamples were collected and serum was prepared to determine the level ofhAGT mRNA expression relative to the prebleed level of hAGT. The resultsof these experiments are shown in Table 14.

TABLE 14 hAGT single dose dose-response screen in hAGT expressing miceDose Day 0 Day 3 Day 7 Day 14 Duplex mpk Avg SD Avg SD Avg SD Avg SD PBS1.00 0.00 1.29 0.28 0.91 0.17 0.65 0.05 AD- 3 1.00 0.00 0.42 0.09 0.160.04 0.11 0.04 67327 1 1.00 0.00 0.63 0.05 0.44 0.04 0.31 0.06 0.3 1.000.00 1.04 0.13 0.90 0.12 0.77 0.06 0.1 1.00 0.00 0.91 0.06 0.88 0.070.74 0.15 AD- 3 1.00 0.00 0.58 0.14 0.42 0.13 0.37 0.10 67335 1 1.000.00 0.59 0.41 0.64 0.03 0.56 0.12 0.3 1.00 0.00 1.06 0.28 0.81 0.140.80 0.07 0.1 1.00 0.00 1.18 0.18 1.02 0.10 0.85 0.08

Example 5: In Vivo AGT Silencing in a Mouse Expressing hAGT

A series of experiments were performed to assess hAGT target knockdownin mice expressing hAGT from an AAV8 expression vector which has astrong liver tropism. Briefly, mice were infected with 1×10¹¹ AAV8particles containing an expression construct for hAGT under the controlof a constitutive promoter. Two weeks after infection with the AAV8virus, mice were administered a single 1 mg/kg dose of an siRNAtargeting hAGT, or PBS as a control (n=3 per group). The nucleotidesequences of the duplexes assessed in these experiments are provided inTable 15.

TABLE 15Modified Sense and Antisense Strand Sequences of AGT dsRNAs (21/23 mers)Sense Strand/ SEQ Duplex Location on Antisense Sense Sequence/AntisenseID Parent Name NM_000029.3 Strand Sequence NO. Duplex AD-68577  855-875A-137778 ususccguAfuAfUfAfuggcaugcaaL96 1029 AD-60779  853-875 A-137779usUfsgcaUfgCfCfauauAfuAfcggaasgsc 1030 AD-68581 1741-1761 A-137784ascscugcAfaAfAfAfungagcaauaL96 1031 AD-56029 1739-1761 A-137785usAfsuugCfuCfAfauunUfuGfcaggususc 1032 AD-68582 1741-1761 A-137784ascscugcAfaAfAfAfungagcaauaL96 1033 AD-56029 1739-1761 A-137786usAfsuugCfucaauuuUfuGfcaggususc 1034 AD-67335 1894-1914 A-134536usgsuuugCfuGfUfGfuaugaucaaaL96 1035 AD-60781 1892-1914 A-134538usUfsugaUfcAfUfacacAfgCfaaacasgsg 1036 AD-67327 2081-2101 A-134527uscsucccAfcCfUfUfuucuucuaauL96 1037 AD-52474 2079-2101 A-134528asUfsuagAfagaaaagGfuGfggagascsu 1038 AD-68575 2084-2104 A-137775cscscaccUfuUfUfCfuucuaaugaaL96 1039 AD-60777 2082-2104 A-137776usUfscauUfaGfAfagaaAfaGfgugggsasg 1040 AD-68576 2084-2104 A-137775cscscaccUfuUfUfCfuucuaaugaaL96 1041 AD-60777 2082-2104 A-137777usUfscauUfagaagaaAfaGfgugggsasg 1042 AD-68583 2291-2311 A-137787cscsagcuUfgUfUfUfgugaaacaaaL96 1043 AD-56019 2289-2311 A-137788usUfsuguUfuCfAfcaaaCfaAfgcuggsusc 1044 AD-68584 2291-2311 A-137787cscsagcuUfgUfUfUfgugaaacaaaL96 1045 AD-56019 2289-2311 A-137789usUfsuguUfucacaaaCfaAfgcuggsusc 1046 AD-67313 2309-2329 A-134509asasaaaaGfuGfUfUfcccuuuucaaL96 1047 AD-60776 2307-2329 A-134510usUfsgaaAfagggaacAfcUfuuuuusgsu 1048 AD-67314 2309-2329 A-134509asasaaaaGfuGfUfUfcccuuuucaaL96 1049 AD-60776 2307-2329 A-134511usUfsgaaAfaGfGfgaacAfcUfuuuuusgsu 1050

Seven days after administration of the agents, blood samples werecollected and serum was prepared to determine the level of hAGT mRNAexpression relative to the prebleed level of hAGT. The results of theseexperiments are provided in Table 16.

TABLE 16 hAGT single dose dose-response screen in hAGT expressing miceDuplex Average SD PBS 98.6 8.7 AD-68577 65.1 7.7 AD-68581 53.1 3.3AD-68582 44.8 11.1 AD-67335 60.5 3.1 AD-67327 43.0 1.0 AD-68575 48.0 9.4AD-68576 47.4 5.9 AD-68583 51.3 7.7 AD-68584 61.0 2.1 AD-67313 70.8 7.6AD-67314 66.2 5.7

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A double-stranded ribonucleic acid (RNAi) agent for inhibitingexpression of angiotensinogen (AGT) in a cell, wherein saiddouble-stranded RNAi agent comprises a sense strand and an antisensestrand forming a double-stranded region, wherein said sense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:1, and saidantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of SEQ IDNO:2, wherein substantially all of the nucleotides of said sense strandand substantially all of the nucleotides of said antisense strand aremodified nucleotides, and wherein said sense strand is conjugated to aligand attached at the 3′-terminus.
 2. The double-stranded ribonucleicacid RNAi agent of claim 1, wherein the sense strand comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromnucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875;847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624;1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360;2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333-2360;2333-2358; 485-503; 517-535; 560-578; 635-653; 803-821; 814-832;822-840; 825-843; 834-852; 837-855; 841-859; 855-873; 967-985;1247-1265; 1248-1266; 1249-1267; 1251-1269; 1253-1271; 1566-1584;1570-1588; 1572-1590; 1574-1592; 1584-1602; 1587-1605; 1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747;1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845;1880-1989; 1892-1914; 1894-1914; 1894-2012; 2035-2053; 2046-2064;2057-2075; 2070-2088; 2072-2090; 2078-2096; 2078-2107; 2078-2011;2080-2098; 2081-2099; 2081-2104; 2081-2011; 2082-2100; 2084-2102;2084-2011; 2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143;2167-2185; 2179-2197; 2201-2219; 2202-2220; 2203-2221; 2204-2222;2227-2245; 2230-2248; 2234-2252; 2244-2264; 2255-2273; 2266-2284;2268-2286; 2270-2288; 2279-2297; 2281-2299; 2283-2301; 2284-2302;2285-2303; 2286-2304; 2288-2306; 2290-2308; 2291-2309; 2291-2311;2291-2318; 2291-2315; 2292-2310; 2294-2312; 2296-2314; 2299-2317;2304-2322; 2304-2329; 2306-2324; 2307-2325; 2309-2327; 2309-2329;2309-2342; 2309-2350; 2309-2358; 2314-2332; 2316-2334; 2317-2335;2320-2338; 2321-2339; 2323-2341; 2325-2343; 2326-2344; 2328-2346;2329-2347; 2331-2349; 2333-2351; 2334-2352; 2335-2353; 2339-2357;2340-2358; or 2341-2359 of the nucleotide sequence of SEQ ID NO:1 andthe antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotides at thecorresponding position of the nucleotide sequence of SEQ ID NO:2, andwherein the antisense strand is substantially complementary to the sensestrand. 3.-6. (canceled)
 7. The double-stranded RNAi agent of claim 1,wherein at least one of said modified nucleotides is selected from thegroup consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an unlockednucleotide, a conformationally restricted nucleotide, a constrainedethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide,a 2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or a dodecanoic acid bisdecylamide group. 8.The double-stranded RNAi agent of claim 1, wherein at least one strandcomprises a 3′ overhang of at least 1 nucleotide; or at least one strandcomprises a 3′ overhang of at least 2 nucleotides.
 9. (canceled)
 10. Adouble-stranded ribonucleic acid (RNAi) agent capable of inhibiting theexpression of angiotensinogen (AGT) in a cell, wherein saiddouble-stranded RNAi agent comprises a sense strand and an antisensestrand forming a double-stranded region, wherein said antisense strandcomprises a region complementary to part of an mRNA encoding AGT,wherein each strand is about 14 to about 30 nucleotides in length,wherein said double-stranded RNAi agent is represented by formula (III):sense: 5′n _(p)—N_(a)—(X X X)_(i)—N_(b)—Y Y Y—N_(b)—(Z Z Z)_(j)—N_(a)-n_(q)3′antisense: 3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III) wherein: i, j, k, and 1 are each independently 0 or 1; p,p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 0-25nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; each n_(p), n_(o)′,n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides; modificationson N_(b) differ from the modification on Y and modifications on N_(b)′differ from the modification on Y′; and wherein the sense strand isconjugated to at least one ligand.
 11. The double-stranded RNAi agent ofclaim 10, wherein i is 0; j is 0; i is 1; j is 1; both i and j are 0;both i and j are 1; k is 0; 1 is 0; k is 1; l is 1; both k and l are 0;or both k and l are
 1. 12. (canceled)
 13. The double-stranded RNAi agentof claim 10, wherein XXX is complementary to X′X′X′, YYY iscomplementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.
 14. Thedouble-stranded RNAi agent of claim 10, wherein the YYY motif occurs ator near the cleavage site of the sense strand. 15.-20. (canceled) 21.The double-stranded RNAi agent of claim 1, wherein the double-strandedregion is 15-30 nucleotide pairs in length; 17-23 nucleotide pairs inlength; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs inlength; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs inlength. 22.-26. (canceled)
 27. The double-stranded RNAi agent of claim1, wherein each strand is independently 15-30 nucleotides in length; or19-30 nucleotides in length. 28.-30. (canceled)
 31. The double-strandedRNAi agent of claim 1, wherein the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker.32. The double-stranded RNAi agent of claim 1, wherein the ligand is


33. The double-stranded RNAi agent of claim 1, wherein the ligand isattached to the 3′ end of the sense strand.
 34. The double-stranded RNAiagent of claim 33, wherein the RNAi agent is conjugated to the ligand asshown in the following schematic

wherein X is O or S.
 35. The double-stranded RNAi agent of claim 1,wherein said RNAi agent further comprises at least one phosphorothioateor methylphosphonate internucleotide linkage. 36.-52. (canceled)
 53. Thedouble-stranded RNAi agent of claim 1, wherein the sense strand is 21nucleotides in length and the antisense strand is 23 nucleotides inlength.
 54. (canceled)
 55. (canceled)
 56. The double-stranded RNAi agentof claim 1, wherein said RNAi agent is selected from the group of RNAiagents listed in any one of Tables 3, 4, 7, 8, 11, 13, and
 15. 57.-67.(canceled)
 68. A cell containing the double-stranded RNAi agent ofclaim
 1. 69. A pharmaceutical composition comprising the double-strandedRNAi agent of claim
 1. 70.-74. (canceled)
 75. A method of inhibitingangiotensinogen (AGT) expression in a cell, the method comprising: (a)contacting the cell with the double-stranded RNAi agent of claim 1, orthe pharmaceutical composition of claim 69; and (b) maintaining the cellproduced in step (a) for a time sufficient to obtain degradation of themRNA transcript of a AGT gene, thereby inhibiting expression of the AGTgene in the cell. 76.-79. (canceled)
 80. A method of treating a subjecthaving a angiotensinogen (AGT)-associated disorder, comprisingadministering to the subject a therapeutically effective amount of thedouble-stranded RNAi agent of claim 1, or the pharmaceutical compositionof claim 69, thereby treating said subject. 81.-83. (canceled)
 84. Themethod of claim 80, wherein the subject is a human.
 85. The method ofclaim 84, wherein the angiotensinogen-associated disease is selectedfrom the group consisting of hypertension, borderline hypertension,primary hypertension, secondary hypertension, hypertensive emergency,hypertensive urgency, isolated systolic or diastolic hypertension,pregnancy-associated hypertension, diabetic hypertension, resistanthypertension, refractory hypertension, paroxysmal hypertension,renovascular hypertension, Goldblatt hypertension, ocular hypertension,glaucoma, pulmonary hypertension, portal hypertension, systemic venoushypertension, systolic hypertension, labile hypertension; hypertensiveheart disease, hypertensive nephropathy, atherosclerosis,arteriosclerosis, vasculopathy, diabetic nephropathy, diabeticretinopathy, chronic heart failure, cardiomyopathy, diabetic cardiacmyopathy, glomerulosclerosis, coarctation of the aorta, aortic aneurism,ventricular fibrosis, Cushing's syndrome, and other glucocorticoidexcess states including chronic steroid therapy, pheochromocytoma,reninoma, secondary aldosteronism and other mineralocorticoid excessstates, sleep apnea, thyroid/parathyroid disease, heart failure,myocardial infarction, angina, stroke, diabetes mellitus, renal disease,renal failure, systemic sclerosis, intrauterine growth restriction(IUGR), and fetal growth restriction. 86.-101. (canceled)